Implants and biodegradable tissue markers

ABSTRACT

Implantable materials may be used in an iatrogenic site. Applications include radioopaque materials for fiducial marking.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/296,795, filed Mar. 8, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/613,555, filed Jun. 5, 2017, which is acontinuation of U.S. patent application Ser. No. 15/066,707, filed Mar.10, 2016, which is a continuation of U.S. patent application Ser. No.14/465,202, filed Aug. 21, 2014, which is a divisional of U.S. patentapplication Ser. No. 13/750,570, filed Jan. 25, 2013, which is adivisional of U.S. patent application Ser. No. 12/968,527, filed Dec.15, 2010, which claims the benefit of U.S. Provisional Application No.61/286,450, filed Dec. 15, 2009, each of which are hereby incorporatedby reference herein in their entirety.

TECHNICAL FIELD

The technical field, in general, relates to stabilizing and visualizingtissue gaps left by surgical removal of cancerous tissues; certainembodiments include polymeric microparticles with attached radioopaquemarkers.

BACKGROUND

According to the American Cancer Society, in 2009 it is estimated therewill be more than 192,000 and 62,000 new cases of invasive and in situbreast cancer, respectively, with more with over 40,000 deaths in theUnited States alone. Once detected, most breast cancers, includingductal carcinoma in situ (DCIS), are removed surgically either by amodified radical mastectomy, or via lumpectomy. Following lumpectomy,patents are then typically treated with either chemotherapy followed by5-7 weeks of whole breast external beam radiation therapy (EBRT), or by5-7 days of accelerated partial breast irradiation (APBI) followed byeither chemotherapy or no further treatment.

SUMMARY

Implants are described herein that conformally fill surgical sites.Conformal filling of the sites with a radioopaque material provides forlater identification and monitoring of the site and its tissue margins.Good visualization allows for careful post-operative follow-up of cancerpatients who have had cancerous tissue removed. In the first place,filling substantially all of the site provides a bulky mass that resistspermanent deformation and migration of the margins. Further, the marginscan be visualized because the site is substantially full and the implantis thus coterminous with the tissue margins.

One embodiment of an implant involves filling a site with flowableprecursors that set-up to make a hydrogel implant that provides forready visualization of margins of the implant site. The implantimmobilizes and may adhere to the tissue edges, so that the edges can befollowed and subsequently treated. A process for making the implantinvolves reacting precursors with each other that form the implant whenthey react with each other. A crosslinked hydrogel can be formed in-situthat supports tissue around a lumpectomy site to stabilize the tissue atthe margins of the lumpectomy so the margins can be precisely targetedby subsequent treatments, for instance, radiation or ablation.

Another embodiment of the invention provides filling the site with smallparticles that are small, pliable, and slippery so that they flow easilyinto the site and its irregularities, pack closely, and provide goodvisualization of the margins. Radioopaque agents may be included withthe implants, either covalently attached or mixed within the materials.

A conformal filling approach is a considerable improvement over the useof clips, which provide poor resolution of the site's margins. Conformalfilling also improves over a do-nothing approach which is also aconventional practice that allows a void to remain at the surgical siteto be filled with a seroma. Seromas can be symptomatic, often requiringdrainage, and are known to change size following surgery, preventingtargeting for partial breast irradiation. The implants may be formulatedto be stable until no longer needed, and then biodegrade. The implantsmay also be used with or without radioopaque agents. An in situ formedhydrogel can seal tissue margins to reduce seroma formation. Further,the use of hydrogel as a continuous phase or as a particulate form mayresult in improved cosmesis since the hydrogel fills the cavity andprevents its deformation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of the prior art for removing a tumor from atissue.

FIG. 1B is an illustration of the prior art for removing a tumor from atissue.

FIG. 2A illustrates placement of matrix precursors in an iatrogenic siteusing a dual-barreled applicator.

FIG. 2B depicts an alternative applicator for placing a plurality ofparticles into the site of FIG. 2A.

FIG. 3A is an image of a hydrogel placed in an iatrogenic site withclear definition of the lumpectomy cavity on kilovoltage CT.

FIG. 3B is a T2-weighted MRI image of the site of FIG. 3A.

FIG. 3C is a kilovoltage cone-beam CT image of the site of FIG. 3A.

FIG. 3D is a photomicrograph image of a gross axial section (hydrogel isdyed blue) of the site of FIG. 3A.

FIG. 3E is an axial ultrasound image of the site of FIG. 3A.

FIG. 4A is a CT image of a hydrogel iatrogenic site treatments of a 63cc lumpectomy that was filled with an equal volume of hydrogel.

FIG. 4B is an MRI image of the site of FIG. 4A.

FIG. 4C is a CT image of a 31 cc lumpectomy site that was partiallyfilled with 18 cc of hydrogel.

FIG. 4D is an MRI image of the site of FIG. 4C.

FIG. 4E is a 33 cc lumpectomy cavity site that was sutured closed (thesuperior and inferior cavity walls were apposed) and then injected with18 cc of hydrogel; the hydrogel marks the edges of the cavity, outliningthe apposed tissue.

FIG. 4F is an MRI image of the site of FIG. 4E.

FIG. 5A provides details of five-field, partial-breast radiationtreatment plans generated before a hydrogel was injected into lumpectomycavities. Axial sections are shown. 105%, 95%, 50%, and 30% isodosecontours are shown in lines 105′, 95′, 50′, and 30′, respectively.

FIG. 5B is a radiation treatment plan generated after the hydrogelinjection of FIG. 5A.

FIG. 5C is a DVH that shows only slightly higher radiation doses to thenormal breast tissue, ipsilateral lung, and heart in the posthydrogelplan (dashed lines).

FIG. 6A is a plot of breast (non-PTV) V50% for a set of treatment planswith 25 mm margins. Each line corresponds to one pair ofpre/post-hydrogel plans. The breast (non-PTV) V50% increased due tohydrogel placement in four of five cases; however, the increases weremodest compared with the volume constraint of 50%.

FIG. 6B is a plot of the treatment plans of FIG. 6A and shows howipsilateral lung V30% also increased in four of five cases; increaseswere more sizeable relative to the volume constraint of 15%.

FIG. 6C is a plot of the treatment plans of FIG. 7A and shows how, forall left sided lumpectomies, hydrogel increased the heart V5%, but thevolumes remained well under the 40% constraint.

FIG. 7A is a plot that describes treatment plans with 15 mm margins. Thebreast (non-PTV) V50% decreased in all five cases.

FIG. 7B is a plot of the treatment plans of FIG. 7A and shows howipsilateral lung V30% also decreased in four of five cases.

FIG. 7C is a plot of the treatment plans of FIG. 7A and shows how, forall left-sided lumpectomies, the heart V5% also showed small decreases.

FIG. 8 is a plot of radiopacity as a function of iodine concentration,with an iodinated polyethylene glycol (PEG-I), potassium iodide (KI) andiodine (OMNIPAQUE) being compared.

FIG. 9 is a plot depicting radiopacity of samples including hydrogelswith bound iodine over time (Example 4), without correction for hydrogelswelling. The first percentage indicates the iodine (as TIB)substitution as a percentage of multi-armed precursor arms and thesecond percentage indicates the solids content of the hydrogel as apercent.

FIG. 10 is a plot depicting radiopacity of the samples of FIG. 9, with acorrection for hydrogel swelling.

FIG. 11A shows how an osmotic agent may be used to reduce the forcerequired to eject a collection of particles from a small gauge needle.

FIG. 11B depicts shrinkage of hydrogels in the presence of the osmoticagents of FIG. 11A.

DETAILED DESCRIPTION

Tissues around an iatrogenic (medically-created) site can be stabilizedby conformal implants that are placed in the site. The implants'conforming to the sites provides for accurate follow-on treatments.After removal of malignant tissue material, for instance, furthertreatment of tissue around the removed material is often desired, forinstance, by radiation of the margins of the site or tissue-ablativetechniques. It is difficult to target the margins with precision,however, since the margins are hard to visualize and shift size andshape over the course of time. FIGS. 1A and 1B depict breast tissue 100having tumor 102. Surgery involves removal of tumor 102 and surroundingtissue. The removed tissue material 104 has a shape and a volume.Removal of material 104 creates iatrogenic site 110 (also referred to asa cavity) also having a shape and a volume that are defined by surface116, which is the tissue margins of the iatrogenic site. Site 110 isclearly bounded, with those boundaries being surfaces. In the case of asite that is not entirely within the body, the shape and volume of thesite can nonetheless be defined with reasonable accuracy by referringthe shape and volume of the removed material.

Conventionally, the cavity is imaged before radiation. In cases wherethere is no seroma, the cavity is hard to even identify. While clips forimaging are helpful, these provide only individual points that do notdefine the cavity edges. Even when a seroma is present, the cavitychanges shape over the several weeks of radiation. The target thuschanges from the initial plan, potentially moving cancer cells out ofthe radiation, or healthy tissue into it. As a result, it is sometimesnecessary to simply irradiate the whole breast.

One embodiment of the invention solves these problems by providing amatrix that conformally fills the site where it is placed. Experimentshave shown that it is possible to have good indirect visibility and trueconformal filling with this approach, and that such a hydrogel mayadditionally and/or alternatively be used as a fiducial marker. Thehydrogel can be degradable over a span of time that provides stabilityduring a medically required time but dissolution afterwards. Thestability of the hydrogel provides for stability of the site, whichmight otherwise change shape. FIG. 2A schematically depicts filling site110 using a double-barreled applicator that supplies precursors thatform the hydrogel inside site 110. FIG. 2B depicts an alternativesyringe applicator 122 loaded with a plurality of particles 124 that areplaced in the site to form a matrix. Catheters and other applicators mayalso be used.

Example 1 describes a matrix that is applied to an iatrogenic site as aliquid mixture of two precursors that covalently crosslink with eachother to form a hydrogel. The hydrogel revealed fine details of thelumpectomy cavity shape and size, with good correspondence between CT,MRI, cone-beam CT, and gross pathologic sections. Importantly, thelumpectomy site was well defined for partially filled cavities and evenfor cavities that were sutured closed (which, historically, have beenvery difficult to accurately define). In all cases, gross dissectionshowed that hydrogel coated the entire cavity surface, i.e., was trulyconformal to the tissue margins of the site. FIG. 3 shows that thehydrogel clearly defined the site: kilovoltage CT in FIG. 3A;T2-weighted MRI in FIG. 3B; kilovoltage cone-beam CT in FIG. 3C; grossaxial section (hydrogel is dyed blue) in FIG. 3D, and axial ultrasoundin FIG. 3E. FIG. 4 shows good definition with CT and MRI: a 63 cc volumedefect site was filled with an equal volume of hydrogel (FIG. 4A (CT)and FIG. 4B (MRI)). In FIG. 4C (CT) and FIG. 4D (MRI), a 31 cclumpectomy was partially filled with 18 cc of hydrogel, and shows thatthe cavity is still well defined. In FIG. 4E (CT) and FIG. 4F (MRI) a 33cc lumpectomy cavity was sutured closed and then injected with 18 cc ofhydrogel; the hydrogel marks the edges of the cavity, outlining theapposed tissue.

Example 2 describes a series of radiation plans for the treated sites,and compares plans for hydrogel-filled versus not-filled sites.Radiation plans are routinely made, and consider how much radiation toapply to a site in light of various considerations such as the desireddose, treatment regimen, and radiation limits for nearby healthytissues. It is contrary to conventional wisdom to expand iatrogenicsites because it is well known that increased target size can increaseradiation doses to nearby, normal tissues.

In fact, this undesirable effect was observed with the particularhydrogel that was tested, as illustrated in FIGS. 5 and 6 (see detailsin Example 2). FIG. 6 shows five-field, partial-breast radiationtreatment plans generated both before and after the hydrogel wasinjected into the cavities. Axial sections (prehydrogel in FIG. 5A andpost-hydrogel in FIG. 5B) are shown. 105%, 95%, 50%, and 30% isodosecontours are shown in 105′, 95′, 50′, 30′, respectively. Theevaluation-PTV is shaded and lies substantially in the 95′ area. The DVH(FIG. 5C) shows slightly higher radiation doses to the normal breasttissue, ipsilateral lung, and heart in the posthydrogel plan (dashedlines). Evaluation-PTV coverage is similar, although the post-hydrogelplan shows slightly increased inhomogeneity. Similarly, FIG. 6 showsthat, when using standard treatment expansion (25 mm), the hydrogelimplant tends to increase normal tissues doses. Each line corresponds toone pair of pre/post-hydrogel plans. The breast (non-PTV) V50% increaseddue to hydrogel placement in four of five cases; however, the increaseswere modest compared with the volume constraint of 50% (FIG. 6A).Ipsilateral lung V30% also increased in four of five cases; increaseswere more sizeable relative to the volume constraint of 15% (FIG. 6B).For the left sided lumpectomies, hydrogel increased the heart V5%, butthe volumes remained well under the 40% constraint (FIG. 6C).

Conventional treatment expansions of 25 mm beyond the PTV are utilized,in part, because if poor visualization of the cavity margins during doseplanning. If the hydrogel was effective at delineating the cavitymargins, then treatment localization uncertainty decreased, and atreatment expansion of 15 mm beyond the PTV may well be feasible. With atreatment expansion of 15 mm radiation exposure of healthy surroundingtissues was reduced compared to conventional treatments. FIG. 7 (seeExample 2) details this effect. The breast (non-PTV) V50% decreased inall five cases (FIG. 7A). Ipsilateral lung V30% also decreased in fourof five cases (FIG. 7B). For the left-sided lumpectomies, the heart V5%also showed small decreases (FIG. 7C). Consideration of the superiorvisualization aspects of the implant points to methods involving use ofa hydrogel as an implant at a iatrogenic site with reduced treatmentexpansions, e.g., expansions of less than about 25 mm, or between about2 and about 25 mm; artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated,e.g., less than about 20 mm, or from about 5 mm to less than about 25mm.

Hydrogels and Hydrogel Precursors

Accordingly, embodiments are provided herein for making implantmaterials. Such materials include matrices with a porosity of more thanabout 20% v/v; artisans will immediately appreciate that all the rangesand values within the explicitly stated range is contemplated. Hydrogelsare an embodiment of such an implant. Hydrogels are materials that donot dissolve in water and retain a significant fraction (more than 20%)of water within their structure. In fact, water contents in excess of90% are often known. Hydrogels are often formed by crosslinking watersoluble molecules to form networks of essentially infinite molecularweight. Hydrogels with high water contents are typically soft, pliablematerials. A hydrogel that has been dried is referred to herein as adehydrated hydrogel if it will return to a hydrogel state upon exposureto water; this hydrogel would expand in volume if it were exposed to anexcess of water and not constrained. The term desiccated refers to ahydrogel essentially having no fluids, bearing in mind that some traceamounts of water may nonetheless be present.

Hydrogels may be formed from natural, synthetic, or biosyntheticpolymers. Natural polymers may include glycosminoglycans,polysaccharides, and proteins. Some examples of glycosaminoglycansinclude dermatan sulfate, hyaluronic acid, the chondroitin sulfates,chitin, heparin, keratan sulfate, keratosulfate, and derivativesthereof. In general, the glycosaminoglycans are extracted from a naturalsource and purified and derivatized. However, they also may besynthetically produced or synthesized by modified microorganisms such asbacteria. These materials may be modified synthetically from a naturallysoluble state to a partially soluble or water swellable or hydrogelstate. This modification may be accomplished by various well-knowntechniques, such as by conjugation or replacement of ionizable orhydrogen bondable functional groups such as carboxyl and/or hydroxyl oramine groups with other more hydrophobic groups.

For example, carboxyl groups on hyaluronic acid may be esterified byalcohols to decrease the solubility of the hyaluronic acid. Suchprocesses are used by various manufacturers of hyaluronic acid products(such as Genzyme Corp., Cambridge, Mass.) to create hyaluronic acidbased sheets, fibers, and fabrics that form hydrogels. Other naturalpolysaccharides, such as carboxymethyl cellulose or oxidized regeneratedcellulose, natural gum, agar, agrose, sodium alginate, carrageenan,fucoidan, furcellaran, laminaran, hypnea, eucheuma, gum arabic, gumghatti, gum karaya, gum tragacanth, locust beam gum, arbinoglactan,pectin, amylopectin, gelatin, hydrophilic colloids such as carboxymethylcellulose gum or alginate gum cross-linked with a polyol such aspropylene glycol, and the like, also form hydrogels upon contact withaqueous surroundings.

Synthetic hydrogels may be biostable or biodegradable or biodegradable.Examples of bio stable hydrophilic polymeric materials arepoly(hydroxyalkyl methacrylate), poly(electrolyte complexes),poly(vinylacetate) cross-linked with hydrolysable or otherwisedegradable bonds, and water-swellable N-vinyl lactams. Other hydrogelsinclude hydrophilic hydrogels known as CARBOPOL®, an acidic carboxypolymer (Carbomer resins are high molecular weight,allylpentaerythritol-crosslinked, acrylic acid-based polymers, modifiedwith C10-C30 alkyl acrylates), polyacrylamides, polyacrylic acid, starchgraft copolymers, acrylate polymer, ester cross-linked polyglucan. Suchhydrogels are described, for example, in U.S. Pat. No. 3,640,741 toEtes, U.S. Pat. No. 3,865,108 to Hartop, U.S. Pat. No. 3,992,562 toDenzinger et al., U.S. Pat. No. 4,002,173 to Manning et al., U.S. Pat.No. 4,014,335 to Arnold and U.S. Pat. No. 4,207,893 to Michaels, all ofwhich are incorporated herein by reference, with the presentspecification controlling in case of conflict.

Hydrogels may be made from precursors. The precursors are not hydrogelsbut are covalently crosslinked with each other to form a hydrogel andare thereby part of the hydrogel. Crosslinks can be formed by covalentor ionic bonds, by hydrophobic association of precursor moleculesegments, or by crystallization of precursor molecule segments. Theprecursors can be triggered to react to form a crosslinked hydrogel. Theprecursors can be polymerizable and include crosslinkers that are often,but not always, polymerizable precursors. Polymerizable precursors arethus precursors that have functional groups that react with each otherto form polymers made of repeating units. Precursors may be polymers.

Some precursors thus react by chain-growth polymerization, also referredto as addition polymerization, and involve the linking together ofmonomers incorporating double or triple chemical bonds. Theseunsaturated monomers have extra internal bonds which are able to breakand link up with other monomers to form the repeating chain. Monomersare polymerizable molecules with at least one group that reacts withother groups to form a polymer. A macromonomer (or macromer) is apolymer or oligomer that has at least one reactive group, often at theend, which enables it to act as a monomer; each macromonomer molecule isattached to the polymer by reaction the reactive group. Thusmacromonomers with two or more monomers or other functional groups tendto form covalent crosslinks. Addition polymerization is involved in themanufacture of, e.g., polypropylene or polyvinyl chloride. One type ofaddition polymerization is living polymerization.

Some precursors thus react by condensation polymerization that occurswhen monomers bond together through condensation reactions. Typicallythese reactions can be achieved through reacting molecules incorporatingalcohol, amine or carboxylic acid (or other carboxyl derivative)functional groups. When an amine reacts with a carboxylic acid an amideor peptide bond is formed, with the release of water. Some condensationreactions follow a nucleophilic acyl substitution, e.g., as in U.S. Pat.No. 6,958,212, which is hereby incorporated by reference herein in itsentirety to the extent it does not contradict what is explicitlydisclosed herein.

Some precursors react by a chain growth mechanism. Chain growth polymersare defined as polymers formed by the reaction of monomers ormacromonomers with a reactive center. A reactive center is a particularlocation within a chemical compound that is the initiator of a reactionin which the chemical is involved. In chain-growth polymer chemistry,this is also the point of propagation for a growing chain. The reactivecenter is commonly radical, anionic, or cationic in nature, but can alsotake other forms. Chain growth systems include free radicalpolymerization, which involves a process of initiation, propagation andtermination. Initiation is the creation of free radicals necessary forpropagation, as created from radical initiators, e.g., organic peroxidemolecules. Termination occurs when a radical reacts in a way thatprevents further propagation. The most common method of termination isby coupling where two radical species react with each other forming asingle molecule.

Some precursors react by a step growth mechanism, and are polymersformed by the stepwise reaction between functional groups of monomers.Most step growth polymers are also classified as condensation polymers,but not all step growth polymers release condensates.

Monomers may be polymers or small molecules. A polymer is a highmolecular weight molecule formed by combining many smaller molecules(monomers) in a regular pattern. Oligomers are polymers having less thanabout 20 monomeric repeat units. A small molecule generally refers to amolecule that is less than about 2000 Daltons.

The precursors may thus be small molecules, such as acrylic acid orvinyl caprolactam, larger molecules containing polymerizable groups,such as acrylate-capped polyethylene glycol (PEG-diacrylate), or otherpolymers containing ethylenically-unsaturated groups, such as those ofU.S. Pat. No. 4,938,763 to Dunn et al, U.S. Pat. Nos. 5,100,992 and4,826,945 to Cohn et al, or U.S. Pat. Nos. 4,741,872 and 5,160,745 toDeLuca et al., each of which is hereby incorporated by reference hereinin its entirety to the extent it does not contradict what is explicitlydisclosed herein.

To form covalently crosslinked hydrogels, the precursors must becrosslinked together. In general, polymeric precursors will formpolymers that will be joined to other polymeric precursors at two ormore points, with each point being a linkage to the same or differentpolymers. Precursors with at least two reactive groups can serve ascrosslinkers since each reactive group can participate in the formationof a different growing polymer chain. In the case of functional groupswithout a reactive center, among others, crosslinking requires three ormore such functional groups on at least one of the precursor types. Forinstance, many electrophilic-nucleophilic reactions consume theelectrophilic and nucleophilic functional groups so that a thirdfunctional group is needed for the precursor to form a crosslink. Suchprecursors thus may have three or more functional groups and may becrosslinked by precursors with two or more functional groups. Acrosslinked molecule may be crosslinked via an ionic or covalent bond, aphysical force, or other attraction. A covalent crosslink, however, willtypically offer stability and predictability in reactant productarchitecture.

In some embodiments, each precursor is multifunctional, meaning that itcomprises two or more electrophilic or nucleophilic functional groups,such that a nucleophilic functional group on one precursor may reactwith an electrophilic functional group on another precursor to form acovalent bond. At least one of the precursors comprises more than twofunctional groups, so that, as a result of electrophilic-nucleophilicreactions, the precursors combine to form crosslinked polymericproducts.

The precursors may have biologically inert and hydrophilic portions,e.g., a core. In the case of a branched polymer, a core refers to acontiguous portion of a molecule joined to arms that extend from thecore, with the arms having a functional group, which is often at theterminus of the branch. The hydrophilic precursor or precursor portionpreferably has a solubility of at least 1 g/100 mL in an aqueoussolution. A hydrophilic portion may be, for instance, a polyether, forexample, polyalkylene oxides such as polyethylene glycol (PEG),polyethylene oxide (PEO), polyethylene oxide-co-polypropylene oxide(PPO), co-polyethylene oxide block or random copolymers, and polyvinylalcohol (PVA), poly (vinyl pyrrolidinone) (PVP), poly (amino acids,dextran, or a protein. The precursors may have a polyalkylene glycolportion and may be polyethylene glycol based, with at least about 80% or90% by weight of the polymer comprising polyethylene oxide repeats. Thepolyethers and more particularly poly (oxyalkylenes) or poly (ethyleneglycol) or polyethylene glycol are generally hydrophilic.

A precursor may also be a macromolecule (or macromer), which is amolecule having a molecular weight in the range of a thousand to manymillions. In some embodiments, however, at least one of the precursorsis a small molecule of about 1000 Da or less. The macromolecule, whenreacted in combination with a small molecule of about 1000 Da or less,is preferably at least five to fifty times greater in molecular weightthan the small molecule and is preferably less than about 60,000 Da;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated. A more preferredrange is a macromolecule that is about seven to about thirty timesgreater in molecular weight than the crosslinker and a most preferredrange is about ten to twenty times difference in weight. Further, amacromolecular molecular weight of 5,000 to 50,000 is useful, as is amolecular weight of 7,000 to 40,000 or a molecular weight of 10,000 to20,000.

Certain macromeric precursors are the crosslinkable, biodegradable,water-soluble macromers described in U.S. Pat. No. 5,410,016 to Hubbellet al, which is hereby incorporated herein by reference in its entiretyto the extent it does not contradict what is explicitly disclosed. Thesemacromers are characterized by having at least two polymerizable groups,separated by at least one degradable region.

Synthetic precursors may be used. Synthetic refers to a molecule notfound in nature or not normally found in a human. Some syntheticprecursors are free of amino acids or free of amino acid sequences thatoccur in nature. Some synthetic precursors are polypeptides that are notfound in nature or are not normally found in a human body, e.g., di-,tri-, or tetra-lysine. Some synthetic molecules have amino acid residuesbut only have one, two, or three that are contiguous, with the aminoacids or clusters thereof being separated by non-natural polymers orgroups. Polysaccharides or their derivatives are thus not synthetic.

Alternatively, natural proteins or polysaccharides may be adapted foruse with these methods, e.g., collagens, fibrin(ogen)s, albumins,alginates, hyaluronic acid, and heparins. These natural molecules mayfurther include chemical derivitization, e.g., synthetic polymerdecorations. The natural molecule may be crosslinked via its nativenucleophiles or after it is derivatized with functional groups, e.g., asin U.S. Pat. Nos. 5,304,595, 5,324,775, 6,371,975, and 7,129,210, eachof which is hereby incorporated by reference to the extent it does notcontradict what is explicitly disclosed herein. Natural refers to amolecule found in nature. Natural polymers, for example proteins orglycosaminoglycans, e.g., collagen, fibrinogen, albumin, and fibrin, maybe crosslinked using reactive precursor species with electrophilicfunctional groups. Natural polymers normally found in the body areproteolytically degraded by proteases present in the body. Such polymersmay be reacted via functional groups such as amines, thiols, orcarboxyls on their amino acids or derivatized to have activatablefunctional groups. While natural polymers may be used in hydrogels,their time to gelation and ultimate mechanical properties must becontrolled by appropriate introduction of additional functional groupsand selection of suitable reaction conditions, e.g., pH.

Precursors may be made with a hydrophobic portion provided that theresultant hydrogel retains the requisite amount of water, e.g., at leastabout 20%. In some cases, the precursor is nonetheless soluble in waterbecause it also has a hydrophilic portion. In other cases, the precursormakes dispersion in the water (a suspension) but is nonethelessreactable to from a crosslinked material. Some hydrophobic portions mayinclude a plurality of alkyls, polypropylenes, alkyl chains, or othergroups. Some precursors with hydrophobic portions are sold under thetrade names PLURONIC F68, JEFFAMINE, or TECTRONIC. A hydrophobic portionis one that is sufficiently hydrophobic to cause the macromer orcopolymer to aggregate to form micelles in an aqueous continuous phaseor one that, when tested by itself, is sufficiently hydrophobic toprecipitate from, or otherwise change phase while within, an aqueoussolution of water at pH from about 7 to about 7.5 at temperatures fromabout 30 to about 50 degrees Centigrade.

Precursors may have, e.g., 2-100 arms, with each arm having a terminus,bearing in mind that some precursors may be dendrimers or other highlybranched materials. An arm on a hydrogel precursor refers to a linearchain of chemical groups that connect a crosslinkable functional groupto a polymer core. Some embodiments are precursors with between 3 and300 arms; artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated, e.g., 4 to16, 8 to 100, or at least 6 arms.

Thus hydrogels can be made, e.g., from a multi-armed precursor with afirst set of functional groups and a low molecular-weight precursorhaving a second set of functional groups. For example, a six-armed oreight-armed precursor may have hydrophilic arms, e.g., polyethyleneglycol, terminated with primary amines, with the molecular weight of thearms being about 1,000 to about 40,000; artisans will immediatelyappreciate that all ranges and values within the explicitly statedbounds are contemplated. Such precursors may be mixed with relativelysmaller precursors, for example, molecules with a molecular weight ofbetween about 100 and about 5000, or no more than about 800, 1000, 2000,or 5000 having at least about three functional groups, or between about3 to about 16 functional groups; ordinary artisans will appreciate thatall ranges and values between these explicitly articulated values arecontemplated. Such small molecules may be polymers or non-polymers andnatural or synthetic.

Precursors that are not dendrimers may be used. Dendritic molecules arehighly branched radially symmetrical polymers in which the atoms arearranged in many arms and subarms radiating out from a central core.Dendrimers are characterized by their degree of structural perfection asbased on the evaluation of both symmetry and polydispersity and requireparticular chemical processes to synthesize. Accordingly, an artisan canreadily distinguish dendrimer precursors from non-dendrimer precursors.Dendrimers have a shape that is typically dependent on the solubility ofits component polymers in a given environment, and can changesubstantially according to the solvent or solutes around it, e.g.,changes in temperature, pH, or ion content.

Precursors may be dendrimers, e.g., as in Patent Application Pub. Nos.US 20040086479, US 20040131582, WO 07005249, WO 07001926, WO 06031358,or the U.S. counterparts thereof; dendrimers may also be useful asmultifunctional precursors, e.g., as in U.S. Pat. Pub. No's. US20040131582, US 20040086479 and PCT Applications No. WO 06031388 and WO06031388; each of which US and PCT applications are hereby incorporatedby reference herein in its entirety to the extent they do not contradictwhat is explicitly disclosed herein. Dendrimers are highly orderedpossess high surface area to volume ratios, and exhibit numerous endgroups for potential functionalization. Embodiments includemultifunctional precursors that are not dendrimers.

Some embodiments include a precursor that consists essentially of anoligopeptide sequence of no more than five residues, e.g., amino acidscomprising at least one amine, thiol, carboxyl, or hydroxyl side chain.A residue is an amino acid, either as occurring in nature or derivatizedthereof. The backbone of such an oligopeptide may be natural orsynthetic. In some embodiments, peptides of two or more amino acids arecombined with a synthetic backbone to make a precursor; certainembodiments of such precursors have a molecular weight in the range ofabout 100 to about 10,000 or about 300 to about 500 Artisans willimmediately appreciate that all ranges and values between theseexplicitly articulated bounds are contemplated.

Precursors may be prepared to be free of amino acid sequences cleavableby enzymes present at the site of introduction, including free ofsequences susceptible to attach by metalloproteinases and/orcollagenases. Further, precursors may be made to be free of all aminoacids, or free of amino acid sequences of more than about 50, 30, 20,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. Precursors may benon-proteins, meaning that they are not a naturally occurring proteinand cannot be made by cleaving a naturally occurring protein and cannotbe made by adding synthetic materials to a protein. Precursors may benon-collagen, non-fibrin, non-fibrinogen), and non-albumin, meaning thatthey are not one of these proteins and are not chemical derivatives ofone of these proteins. The use of non-protein precursors and limited useof amino acid sequences can be helpful for avoiding immune reactions,avoiding unwanted cell recognition, and avoiding the hazards associatedwith using proteins derived from natural sources. Precursors can also benon-saccharides (free of saccharides) or essentially non-saccharides(free of more than about 5% saccharides by w/w of the precursormolecular weight. Thus a precursor may, for example, exclude hyaluronicacid, heparin, or gellan. Precursors can also be both non-proteins andnon-saccharides.

Peptides may be used as precursors. In general, peptides with less thanabout 10 residues are preferred, although larger sequences (e.g.,proteins) may be used. Artisans will immediately appreciate that everyrange and value within these explicit bounds is included, e.g., 1-10,2-9, 3-10, 1, 2, 3, 4, 5, 6, or 7. Some amino acids have nucleophilicgroups (e.g., primary amines or thiols) or groups that can bederivatized as needed to incorporate nucleophilic groups orelectrophilic groups (e.g., carboxyls or hydroxyls). Polyamino acidpolymers generated synthetically are normally considered to be syntheticif they are not found in nature and are engineered not to be identicalto naturally occurring biomolecules.

Some hydrogels are made with a polyethylene glycol-containing precursor.Polyethylene glycol (PEG, also referred to as polyethylene oxide whenoccurring in a high molecular weight) refers to a polymer with a repeatgroup (CH₂CH₂O)_(n), with n being at least 3. A polymeric precursorhaving a polyethylene glycol thus has at least three of these repeatgroups connected to each other in a linear series. The polyethyleneglycol content of a polymer or arm is calculated by adding up all of thepolyethylene glycol groups on the polymer or arm, even if they areinterrupted by other groups. Thus, an arm having at least 1000 MWpolyethylene glycol has enough CH₂CH₂O groups to total at least 1000 MW.As is customary terminology in these arts, a polyethylene glycol polymerdoes not necessarily refer to a molecule that terminates in a hydroxylgroup. Molecular weights are abbreviated in thousands using the symbolk, e.g., with 15K meaning 15,000 molecular weight, i.e., 15,000 Daltons.SG or SGA refers to succinimidyl glutarate. SS refers to succinatesuccinimide. SS and SG are succinimidyl esters that have an ester groupthat degrades by hydrolysis in water. Hydrolytically degradable thusrefers to a material that would spontaneously degrade in vitro in anexcess of water without any enzymes or cells present to mediate thedegradation. A time for degradation refers to effective disappearance ofthe material as judged by the naked eye. Trilysine (also abbreviatedLLL) is a synthetic tripeptide. PEG and/or hydrogels may be provided ina form that is pharmaceutically acceptable, meaning that it is highlypurified and free of contaminants, e.g., pyrogens.

Functional Groups

The precursors have functional groups that react with each other to formthe material, either outside a patient, or in situ. The functionalgroups generally have polymerizable groups for polymerization or reactwith each other in electrophile-nucleophile reactions or are configuredto participate in other polymerization reactions. Various aspects ofpolymerization reactions are discussed in the precursors section herein.

Thus in some embodiments, precursors have a polymerizable group that isactivated by photoinitiation or redox systems as used in thepolymerization arts, e.g., or electrophilic functional groups that arecarbodiimidazole, sulfonyl chloride, chlorocarbonates,n-hydroxysuccinimidyl ester, succinimidyl ester or sulfasuccinimidylesters, or as in U.S. Pat. No. 5,410,016, or 6,149,931, each of whichare hereby incorporated by reference herein in its entirety to theextent they do not contradict what is explicitly disclosed herein. Thenucleophilic functional groups may be, for example, amine, hydroxyl,carboxyl, and thiol. Another class of electrophiles are acyls, e.g., asin U.S. Pat. No. 6,958,212, which describes, among other things, Michaeladdition schemes for reacting polymers.

Certain functional groups, such as alcohols or carboxylic acids, do notnormally react with other functional groups, such as amines, underphysiological conditions (e.g., pH 7.2-11.0, 37° C.). However, suchfunctional groups can be made more reactive by using an activating groupsuch as N-hydroxysuccinimide. Certain activating groups includecarbonyldiimidazole, sulfonyl chloride, aryl halides, sulfosuccinimidylesters, N-hydroxysuccinimidyl ester, succinimidyl ester, epoxide,aldehyde, maleimides, imidoesters and the like. The N-hydroxysuccinimideesters or N-hydroxysulfosuccinimide (NHS) groups are useful groups forcrosslinking of proteins or amine-containing polymers, e.g., aminoterminated polyethylene glycol. An advantage of an NHS-amine reaction isthat the reaction kinetics are favorable, but the gelation rate may beadjusted through pH or concentration. The NHS-amine crosslinkingreaction leads to formation of N-hydroxysuccinimide as a side product.Sulfonated or ethoxylated forms of N-hydroxysuccinimide have arelatively increased solubility in water and hence their rapid clearancefrom the body. An NHS-amine crosslinking reaction may be carried out inaqueous solutions and in the presence of buffers, e.g., phosphate buffer(pH 5.0-7.5), triethanolamine buffer (pH 7.5-9.0), or borate buffer (pH9.0-12), or sodium bicarbonate buffer (pH 9.0-10.0). Aqueous solutionsof NHS based crosslinkers and functional polymers preferably are madejust before the crosslinking reaction due to reaction of NHS groups withwater. The reaction rate of these groups may be delayed by keeping thesesolutions at lower pH (pH 4-7). Buffers may also be included in thehydrogels introduced into a body.

In some embodiments, each precursor comprises only nucleophilic or onlyelectrophilic functional groups, so long as both nucleophilic andelectrophilic precursors are used in the crosslinking reaction. Thus,for example, if a crosslinker has nucleophilic functional groups such asamines, the functional polymer may have electrophilic functional groupssuch as N-hydroxysuccinimides. On the other hand, if a crosslinker haselectrophilic functional groups such as sulfosuccinimides, then thefunctional polymer may have nucleophilic functional groups such asamines or thiols. Thus, functional polymers such as proteins, poly(allylamine), or amine-terminated di- or multifunctional poly(ethylene glycol)can be used.

One embodiment has reactive precursor species with 3 to 16 nucleophilicfunctional groups each and reactive precursor species with 2 to 12electrophilic functional groups each; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated.

The functional groups may be, e.g., electrophiles reactable withnucleophiles, groups reactable with specific nucleophiles, e.g., primaryamines, groups that form amide bonds with materials in the biologicalfluids, groups that form amide bonds with carboxyls, activated-acidfunctional groups, or a combination of the same. The functional groupsmay be, e.g., a strong electrophilic functional group, meaning anelectrophilic functional group that effectively forms a covalent bondwith a primary amine in aqueous solution at pH 9.0 at room temperatureand pressure and/or an electrophilic group that reacts by a ofMichael-type reaction. The strong electrophile may be of a type thatdoes not participate in a Michaels-type reaction or of a type thatparticipates in a Michaels-type reaction.

A Michael-type reaction refers to the 1, 4 addition reaction of anucleophile on a conjugate unsaturated system. The addition mechanismcould be purely polar, or proceed through a radical-like intermediatestate(s); Lewis acids or appropriately designed hydrogen bonding speciescan act as catalysts. The term conjugation can refer both to alternationof carbon-carbon, carbon-heteroatom or heteroatom-heteroatom multiplebonds with single bonds, or to the linking of a functional group to amacromolecule, such as a synthetic polymer or a protein. Michael-typereactions are discussed in detail in U.S. Pat. No. 6,958,212, which ishereby incorporated by reference herein in its entirety for all purposesto the extent it does not contradict what is explicitly disclosedherein.

Examples of strong electrophiles that do not participate in aMichaels-type reaction are: succinimides, succinimidyl esters, orNHS-esters. Examples of Michael-type electrophiles are acrylates,methacrylates, methylmethacrylates, and other unsaturated polymerizablegroups.

Initiating Systems

Some precursors react using initiators. An initiator group is a chemicalgroup capable of initiating a free radical polymerization reaction. Forinstance, it may be present as a separate component, or as a pendentgroup on a precursor. Initiator groups include thermal initiators,photoactivatable initiators, and oxidation-reduction (redox) systems.Long wave UV and visible light photoactivatable initiators include, forexample, ethyl eosin groups, 2, 2-dimethoxy-2-phenyl acetophenonegroups, other acetophenone derivatives, thioxanthone groups,benzophenone groups, and camphorquinone groups. Examples of thermallyreactive initiators include 4, 4′ azobis (4-cyanopentanoic acid) groups,and analogs of benzoyl peroxide groups. Several commercially availablelow temperature free radical initiators, such as V-044, available fromWako Chemicals USA, Inc., Richmond, Va., may be used to initiate freeradical crosslinking reactions at body temperatures to form hydrogelcoatings with the aforementioned monomers.

Metal ions may be used either as an oxidizer or a reductant in redoxinitiating systems. For example, ferrous ions may be used in combinationwith a peroxide or hydroperoxide to initiate polymerization, or as partsof a polymerization system. In this case, the ferrous ions would serveas a reductant. Alternatively, metal ions may serve as an oxidant. Forexample, the ceric ion (4+ valence state of cerium) interacts withvarious organic groups, including carboxylic acids and urethanes, toremove an electron to the metal ion, and leave an initiating radicalbehind on the organic group. In such a system, the metal ion acts as anoxidizer. Potentially suitable metal ions for either role are any of thetransition metal ions, lanthanides and actinides, which have at leasttwo readily accessible oxidation states. Particularly useful metal ionshave at least two states separated by only one difference in charge. Ofthese, the most commonly used are ferric/ferrous; cupric/cuprous;ceric/cerous; cobaltic/cobaltous; vanadate V vs. IV; permanganate; andmanganic/manganous. Peroxygen containing compounds, such as peroxidesand hydroperoxides, including hydrogen peroxide, t-butyl hydroperoxide,t-butyl peroxide, benzoyl peroxide, cumyl peroxide may be used.

An example of an initiating system is the combination of a peroxygencompound in one solution, and a reactive ion, such as a transitionmetal, in another. In this case, no external initiators ofpolymerization are needed and polymerization proceeds spontaneously andwithout application of external energy or use of an external energysource when two complementary reactive functional groups containingmoieties interact at the application site.

Hydrogels and Hydrogel Formation

In general, the precursors may be combined to make acovalently-crosslinked hydrogel. The hydrogel may comprise a therapeuticagent, or agents, released over a suitable period of time. Hydrogels maybe made beforehand or in situ.

When made in situ, the crosslinking reactions generally occur in aqueoussolution under physiological conditions. The crosslinking reactionspreferably do not release heat of polymerization or require exogenousenergy sources for initiation or to trigger polymerization. Formation ofhydrogels in situ can result in adherence of the hydrogel to the tissuemargins. This adherence will tend to reduce fluid flow into the cavityby the bridging of native molecules across the hydrogel barrier andthereby advantageously reduce seroma formation.

The data of Examples 1 and 2 indicates that the hydrogel swelled inplace. An embodiment is a hydrogel with less swelling. The hydrogel maybe generally low-swelling, as measurable by the hydrogel having a weightincreasing no more than about 50% upon exposure to a physiologicalsolution in the absence of physical restraints for twenty-four hoursrelative to a weight of the hydrogel at the time of formation. Swellingmay be measured or expressed by weight or volume. Some embodiments swellby weight or by volume no more than about 50%, no more than about 20%,or no more than about 0%; artisans will immediately appreciate that allthe ranges and values within the explicitly stated ranges arecontemplated, e.g., shrinkage from 10% to 20% (negative swelling),swelling from −10% to no more than 50%. One aspect of swelling is thatlarge changes will increase the difficulty of achieving a desiredhydrogel size. For instance, filling a depression in a tissue with aswelling hydrogel will cause the hydrogel to have a height that is notapparent to the user at the time of application and/or gelation.Similarly, swelling (and shrinkage) can create forces that tend to pullthe hydrogel away from surrounding tissues so that adherence isaffected.

One approach for low-swelling is increase the number of crosslinks orsolids content. Increasing in these factors, however, will typicallyeffect the mechanical properties of the gel, with more crosslinks makingthe gel more brittle but stronger and a higher solids content making thegel stronger. These factors can also increase degradation time and mayaffect interactions with cells. Another embodiment to reduce swelling isto choose precursors that have a high degree of solvation at the time ofcrosslinking but subsequently become less solvated and having a radiusof solvation that effectively shrinks; in other words, the precursor isspread-out in solution when crosslinked but later contracts. Changes topH, temperature, solids concentration, and solvent environment can causesuch changes; moreover, an increase in the number of branches (withother factors being held effectively constant) will tend to also havethis effect. The number of arms are believed to stericly hinder eachother so that they spread-out before crosslinking, but these stericeffects are offset by other factors after polymerization. In someembodiments, precursors have a plurality of similar charges so as toachieve these effects, e.g., a plurality of functional groups having anegative charge, or a plurality of arms each having a positive charge,or each arm having a functional group of similar charges beforecrosslinking or other reaction.

Hydrogels described herein can include hydrogels that swell minimallyafter deposition. Such medical low-swellable hydrogels may have a weightupon polymerization that increases no more than, e.g., about 25%, about10%, about 5%, about 0% by weight upon exposure to a physiologicalsolution, or that shrink (decrease in weight and volume), e.g., by atleast about 5%, at least about 10%, or more. Artisans will immediatelyappreciate that all ranges and values within or otherwise relating tothese explicitly articulated limits are disclosed herein. Unlessotherwise indicated, swelling of a hydrogel relates to its change involume (or weight) between the time of its formation when crosslinkingis effectively complete and the time after being placed in in vitroaqueous solution in an unconstrained state for twenty-four hours, atwhich point it may be reasonably assumed to have achieved itsequilibrium swelling state. For most embodiments, crosslinking iseffectively complete within no more than about three minutes such thatthe initial weight can generally be noted at about 15 minutes afterformation as Weight at initial formation. Accordingly, this formula isused: % swelling=[(Weight at 24 hours−Weight at initialformation)/Weight at initial formation]*100. The weight of the hydrogelincludes the weight of the solution in the hydrogel.

Reaction kinetics are generally controlled in light of the particularfunctional groups, their concentrations, and the local pH unless anexternal initiator or chain transfer agent is required, in which casetriggering the initiator or manipulating the transfer agent can be acontrolling step. In some embodiments, the molecular weights of theprecursors are used to affect reaction times. Precursors with lowermolecular weights tend to speed the reaction due to their higherconcentration of reactive groups, so that some embodiments have at leastone precursor with a molecular weight of less than about 1000 or about2000 Daltons; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated, e.g.,from 100 to about 900 Daltons or from 500 to about 1800 Daltons.

The crosslinking density of the resultant biocompatible crosslinkedpolymer is controlled by the overall molecular weight of the crosslinkerand functional polymer and the number of functional groups available permolecule. A lower molecular weight between crosslinks such as 500 willgive much higher crosslinking density as compared to a higher molecularweight such as 10,000. The crosslinking density also may be controlledby the overall percent solids of the crosslinker and functional polymersolutions. Increasing the percent solids increases the probability thatan electrophilic functional group will combine with a nucleophilicfunctional group prior to inactivation by hydrolysis. Yet another methodto control crosslink density is by adjusting the stoichiometry ofnucleophilic functional groups to electrophilic functional groups. A oneto one ratio leads to the highest crosslink density. Precursors withlonger distances between crosslinks are generally softer, morecompliant, and more elastic. Thus an increased length of a water-solublesegment, such as a polyethylene glycol, tends to enhance elasticity toproduce desirable physical properties. Thus certain embodiments aredirected to precursors with water soluble segments having molecularweights in the range of 3,000 to 100,000; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated e.g., 10,000 to 35,000.

The solids content of the hydrogel can affect its mechanical propertiesand biocompatibility and reflects a balance between competingrequirements. A relatively low solids content is useful, e.g., betweenabout 2.5% to about 20%, including all ranges and values there between,e.g., about 2.5% to about 10%, about 5% to about 15%, or less than about15%.

An embodiment for making a hydrogel in situ in the presence of atherapeutic agent is to combine precursors in an aqueous solution thatcan be administered with an applicator to the punctum and/or canaliculusand thereafter form the hydrogel. The precursors may be mixed with anactivating agent before, during, or after administration. The hydrogelmay be placed with a therapeutic agent dispersed therein, e.g., as asolution, suspension, particles, micelles, or encapsulated.Crosslinking, in one embodiment, entraps the agent. In anotherembodiment, the crosslinking causes the agent to precipitate or movefrom solution to suspension.

Thus one embodiment relates to combining a first hydrogel precursor witha first type of functional groups with a second hydrogel precursorhaving a second type of functional groups in an aqueous solvent in thepresence of a therapeutic agent in the solvent. In one embodiment, theprecursors are dissolved separately and combined in the presence of anactivating agent that provides for effective crosslinking.Alternatively, the mere mixing of the precursors triggers crosslinking.Accordingly, one embodiment is providing branched polymer having aplurality of succinimidyl termini dissolved in a low pH (4.0) diluentsolution) containing a low molecular weight precursor comprisingnucleophiles. This solution is activated by combination with a higher pHsolution (8.8), initiating the crosslinking mechanism. The agent ispre-loaded as a suspension in the diluent solution. The gel forms insitu.

Overview of Other Systems

Certain polymerizable hydrogels made using synthetic precursors areknown in the medical arts, e.g., as used in products such as FOCALSEAL(Genzyme, Inc.), COSEAL (Angiotech Pharmaceuticals), and DURASEAL(Confluent Surgical, Inc.), as in, for example, U.S. Pat. Nos.6,656,200; 5,874,500; 5,543,441; 5,514,379; 5,410,016; 5,162,430;5,324,775; 5,752,974; and 5,550,187; each of which are herebyincorporated by reference to the extent they do not contradict what isexplicitly disclosed herein. These materials can polymerize too quicklyto be injected in a controlled fashion for at least some of theapplications described herein. Also, COSEAL and DURASEAL have a high pH,(above pH 9). Another reason is that they apparently swell too much forfilling of iatrogenic sites. The swelling of COSEAL and DURASEAL hasbeen measured using an in vitro model in comparison to fibrin sealant(Campbell et al., Evaluation of Absorbable Surgical Sealants: In vitroTesting, 2005). Over a three day test, COSEAL swelled an average ofabout 558% by weight, DURASEAL increased an average of about 98% byweight, and fibrin sealant swelled about 3%. Assuming uniform expansionalong all axes, the percent increase in a single axis was calculated tobe 87%, 26%, and 1% for COSEAL, DURASEAL, and fibrin sealantrespectively. FOCALSEAL is known to swell over 300%. And it also needsan external light to be activated. Fibrin sealant is a proteinaceousglue that has adhesive, sealing, and mechanical properties that areinferior to COSEAL, DURASEAL, and other hydrogels disclosed herein.Further, it is typically derived from biological sources that arepotentially contaminated, is cleared from the body by mechanismsdistinct from water-degradation, and typically requires refrigerationwhile stored.

Radioopaque Agents for Hydrogels

Some hydrogel applications would be facilitated if the hydrogel includedradioopaque (RO) agents. These agents may be mixed with the hydrogeland/or covalently attached. One embodiment involves using branchedprecursors that have a covalently attached RO agent, so that thehydrogel will have the RO agent covalently attached upon its formationfrom mixtures of, or including, the RO-labeled precursor.

Examples 3 and 4 demonstrate techniques for incorporation of such agentsinto a matrix. One issue is the need for the RO agent to be present inadequate concentration and volume. The amount of agent that is helpfulcan depend on the tissue site and imaging method. A CT number (alsoreferred to as a Hounsfield unit or number) is a measure of visibilityunder indirect imaging techniques. A CT number was determined forvarious concentrations of the RO agent iohexol, which contains iodine,FIG. 8.

A CT number of at least about 90 may be used. Embodiments includeproviding a matrix (e.g., hydrogel) with a concentration of RO agent togive a CT number of more than about 50; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedrange is contemplated, e.g., at least about 80; about 90 to about 210,or from about 80 to about 2000. Embodiments also include an iodineconcentration between about 0.05% and about 15%; artisans willimmediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated, e.g., from about 0.1% toabout 3%.

For example, an 8-armed PEG precursor of about 10 k Daltons, with 5 ofthe 8 arms terminating in SG and 3 of the 8 arms bound totriiodobenzoate (TIB) at a concentration of 2% gel solids will haveabout 0.18% iodine in the gel and 93 HU, compared to a 20% gel solidsgel with 1.8% iodine in the gel and about 700 HU. 4-arm (Example 3) and8-arm (Example 4) branched precursor molecules had some of their armsbound with TIB, a molecule that contains three iodines. The other PEGarms without TIB were SG functionalized, allowing them to be crosslinked with another precursor (trilysine in the Examples). Thus, theresulting hydrogels had radiopacity from the iodine, FIG. 9. The SGlinkages are hydrolytically labile and thus degrade in water.Persistence of the iodine over a suitable time was addressed bycontrolling the number of functional groups that were derivitized withthe RO agent.

The presence of more than one link to the matrix provides for the ROagent to remain in the matrix and persist until hydrolysis results inthe release and clearance of the precursor-TIB molecules, FIG. 9. Datafrom FIG. 10 shows RO matrices corrected for swell, and indicates iodineretention within the hydrogels. The decrease in RO seen in the 61% TIBsamples is likely due to loss of PEG-TIB molecules, as those gels, withfewer SG linkages, are moving toward complete hydrolysis faster than the31% TIB samples (the 61% TIB-5% has a faster rate than the 61% TIB-10%).The 31% TIB samples, with more SG linkages, appear to have a constantradiopacity, suggesting the loss of PEG-TIB has not started. Takentogether this data suggests that the PEG-TIB linkage certainlywithstands hydrolysis, and a hydrogel made with this linkage would beexpected to retain RO agent, with potential losses due to swelling.

RO agents may be attached to precursors by a variety of methods. Some ofthese methods are set forth in U.S. Pat. No. 7,790,141, which is herebyincorporated by reference herein for all purposes, and including ROagents, precursors, and matrices; in case of conflict, thisspecification controls. Precursors set forth herein and in thisincorporated reference may be decorated with one or more RO agents. Inthe case of a branched or multi-functional precursor, one or more of theavailable reactive sites may be left unreacted. Thus an 8-armedprecursor may have between 1 and 8 functional groups available forcovalent binding to form a matrix and between 1 and 8 functional groupsreplaced by (or reacted with) RO agents. Examples of RO agents aremolecules comprising iodine, TIB, phenyl ring compounds such as 2, 3,5-triiodobenzoic acid, 3, 4, 5-triiodophenol, erythrosine, rose bengal,3, 5-Bis(acetylamino)-2, 4, 6-triiodobenzoic acid, and 3,5-Diacetamido-2, 4, 6-triiodobenzoic acid.

Additional machine-aided imaging agents may be used in addition to, oras alternatives to, radioopaque compounds. Such agents are, for examplefluorescent compounds, ultrasonic contrast agents, or MRI contrastagents (e.g., Gadolinium containing compounds).

Biodegradation

The hydrogel may be made water-degradable, as measurable by the hydrogelbeing dissolvable in vitro in an excess of water by degradation ofwater-degradable groups. This test is predictive ofhydrolytically-driven dissolution in vivo, a process that is in contrastto cell or protease-driven degradation. Significantly, however,polyanhydrides or other conventionally-used degradable materials thatdegrade to acidic components tend to cause inflammation in tissues. Thehydrogels, however, may exclude such materials, and may be free ofpolyanhydrides, anhydride bonds, or precursors that degrade into acid ordiacids.

Instead, for example, SG (succinimidyl glutarate), SS (succinimidylsuccinate), SC (succinimidyl carbonate), SAP (succinimidyl adipate),carboxymethyl hydroxybutyric acid (CM-HBA) may be used and have estericlinkages that are hydrolytically labile. More hydrophobic linkages suchas suberate linkages may also be used, with these linkages being lessdegradable than succinate, glutarate or adipate linkages. Polyethyleneglycols and other precursors may be prepared with these groups. Thecrosslinked hydrogel degradation may proceed by the water-drivenhydrolysis of the biodegradable segment when water-degradable materialsare used. Polymers that include ester linkages may also be included toprovide a desired degradation rate, with groups being added orsubtracted near the esters to increase or decrease the rate ofdegradation. Thus it is possible to construct a hydrogel with a desireddegradation profile, from a few days to many months, using a degradablesegment. If polyglycolate is used as the biodegradable segment, forinstance, a crosslinked polymer could be made to degrade in about 1 toabout 30 days depending on the crosslinking density of the network.Similarly, a polycaprolactone based crosslinked network can be made todegrade in about 1 to about 8 months. The degradation time generallyvaries according to the type of degradable segment used, in thefollowing order: polyglycolate<polylactate<polytrimethylenecarbonate<polycaprolactone. Thus it is possible to construct a hydrogelwith a desired degradation profile, from a few days to many months,using a degradable segment.

The hydrogel may be water-degradable (hydrolytically degradable), asmeasurable by the hydrogel being dissolvable in vitro in an excess ofwater by degradation of water-degradable groups. This test is predictiveof hydrolytically-driven dissolution in vivo, a process that is incontrast to cell or protease-driven degradation. The hydrogels can beselected to be absorbable over days, weeks, or months.

A biodegradable linkage in the hydrogel and/or precursor may bewater-degradable or enzymatically degradable. Illustrativewater-degradable biodegradable linkages include polymers, copolymers andoligomers of glycolide, dl-lactide, 1-lactide, dioxanone, esters,carbonates, and trimethylene carbonate. Illustrative enzymaticallybiodegradable linkages include peptidic linkages cleavable bymetalloproteinases and collagenases. Examples of biodegradable linkagesinclude polymers and copolymers of poly(hydroxy acid)s,poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.

If it is desired that a biocompatible crosslinked matrix bebiodegradable or absorbable, one or more precursors having biodegradablelinkages present in between the functional groups may be used. Thebiodegradable linkage optionally also may serve as the water solublecore of one or more of the precursors used to make the matrix. For eachapproach, biodegradable linkages may be chosen such that the resultingbiodegradable biocompatible crosslinked polymer will degrade or beabsorbed in a desired period of time.

Matrix materials may be chosen so that degradation products are absorbedinto the circulatory system and essentially cleared from the body viarenal filtration. The matrix materials may be hydrogels. One method isto choose precursors that are not broken down in the body, with linkagesbetween the precursors being degraded to return the precursors orprecursors with small changes caused by the covalent crosslinkingprocess. This approach is in contrast to choosing biological matrixmaterials that are destroyed by enzymatic processes and/or materialscleared by macrophages, or that result in by-products that areeffectively not water soluble. Materials that are cleared from the bodyby renal filtration can be labeled and detected in the urine usingtechniques known to artisans. While there might be at least atheoretical loss of some of these materials to other bodily systems, thenormal fate of the material is a kidney clearance process. The termessentially cleared thus refers to materials that are normally clearedthrough the kidneys.

Visualization Agents

A visualization agent may be used with the hydrogel; it reflects oremits light at a wavelength detectable to a human eye so that a userapplying the hydrogel could observe the gel.

Some biocompatible visualization agents are FD&C BLUE #1, FD&C BLUE #2,and methylene blue. These agents are preferably present in the finalelectrophilic-nucleophilic reactive precursor species mix at aconcentration of more than 0.05 mg/ml and preferably in a concentrationrange of at least 0.1 to about 12 mg/ml, and more preferably in therange of 0.1 to 4.0 mg/ml, although greater concentrations maypotentially be used, up to the limit of solubility of the visualizationagent. These concentration ranges can give a color to the hydrogelwithout interfering with crosslinking times (as measured by the time forthe reactive precursor species to gel).

Visualization agents may be selected from among any of the variousnon-toxic colored substances suitable for use in medical implantablemedical devices, such as FD&C BLUE dyes 3 and 6, eosin, methylene blue,indocyanine green, or colored dyes normally found in synthetic surgicalsutures. The visualization agent may be present with either reactiveprecursor species, e.g., a crosslinker or functional polymer solution.The preferred colored substance may or may not become chemically boundto the hydrogel. The visualization agent may be used in smallquantities, e.g., 1% weight/volume, more preferably less that 0.01%weight/volume and most preferably less than 0.001% weight/volumeconcentration; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated.

Drugs or Other Therapeutic Agents for Delivery

The hydrogel or other matrix may be prepared with and used to deliverclasses of drugs including steroids, non-steroidal anti-inflammatorydrugs (NSAIDS), anti-cancer drugs, antibiotics, or others. The hydrogelmay be used to deliver drugs and therapeutic agents, e.g., ananti-inflammatory (e.g., Diclofenac), a pain reliever (e.g.,Bupivacaine), a Calcium channel blocker (e.g., Nifedipine), anAntibiotic (e.g., Ciprofloxacin), a Cell cycle inhibitor (e.g.,Simvastatin), a protein (e.g., Insulin). The rate of release from thehydrogel will depend on the properties of the drug and the hydrogel,with factors including drug sizes, relative hydrophobicities, hydrogeldensity, hydrogel solids content, and the presence of other drugdelivery motifs, e.g., microparticles.

The hydrogel precursor may be used to deliver classes of drugs includingsteroids, NSAIDS, antibiotics, pain relievers, inhibitors or vascularendothelial growth factor (VEGF), chemotherapeutics, antiviral drugs,for instance. The drugs themselves may be small molecules, proteins, RNAfragments, proteins, glycosaminoglycans, carbohydrates, nucleic acid,inorganic and organic biologically active compounds where specificbiologically active agents include but are not limited to: enzymes,antibiotics, antineoplastic agents, local anesthetics, hormones,angiogenic agents, anti-angiogenic agents, growth factors, antibodies,neurotransmitters, psychoactive drugs, anticancer drugs,chemotherapeutic drugs, drugs affecting reproductive organs, genes, andoligonucleotides, or other configurations. The drugs that have low watersolubility may be incorporated, e.g., as particulates or as asuspension. Higher water solubility drugs may be loaded withinmicroparticles or liposomes. Microparticles can be formed from, e.g.,PLGA or fatty acids.

In some embodiments, the therapeutic agent is mixed with the precursorsprior to making the aqueous solution or during the aseptic manufacturingof the functional polymer. This mixture then is mixed with the precursorto produce a crosslinked material in which the biologically activesubstance is entrapped. Functional polymers made from inert polymerslike PLURONIC, TETRONICS or TWEEN surfactants may be used for releasingsmall molecule hydrophobic drugs.

In some embodiments, the therapeutic agent or agents are present in aseparate phase when crosslinker and crosslinkable polymers are reactedto produce a matrix. This phase separation prevents participation ofbioactive substance in the chemical crosslinking reaction such asreaction between NHS ester and amine group. The separate phase also canmodulate the release kinetics of active agent from the crosslinkedmaterial or gel, where ‘separate phase’ could be oil (oil-in wateremulsion), biodegradable vehicle, and the like. Biodegradable vehiclesin which the active agent may be present include: encapsulationvehicles, such as microparticles, microspheres, microbeads,micropellets, and the like, where the active agent is encapsulated in abioerodable or biodegradable polymers such as polymers and copolymersof: poly(anhydride), poly(hydroxy acid)s, poly(lactone)s,poly(trimethylene carbonate), poly(glycolic acid), poly(lactic acid),poly(glycolic acid)-co-poly(glycolic acid), poly(orthocarbonate),poly(caprolactone), crosslinked biodegradable hydrogel networks likefibrin glue or fibrin sealant, caging and entrapping molecules, likecyclodextrin, molecular sieves and the like. Microspheres made frompolymers and copolymers of poly (lactone)s and poly (hydroxy acid) maybe used as biodegradable encapsulation vehicles.

Visualization agents may be included, for instance, in the microspheres,microparticles, and/or microdroplets.

Embodiments of the invention include compositions and methods forforming matrices having entrapped therapeutic compounds. In oneembodiment, a bioactive agent is entrapped in microparticles having ahydrophobic nature (also termed hydrophobic microdomains. In some cases,the resultant composite materials may have two phase dispersions, whereboth phases are absorbable, but are not miscible. For example, thecontinuous phase may be a hydrophilic network (such as a hydrogel, whichmay or may not be crosslinked) while the dispersed phase may behydrophobic (such as an oil, fat, fatty acid, wax, fluorocarbon, orother synthetic or natural water immiscible phase, generically referredto herein as an “oil” or “hydrophobic” phase).

The oil phase entraps the drug and provides a barrier to release bypartitioning of the drug into the hydrogel. The hydrogel phase in turnprotects the oil from digestion by enzymes, such as lipases, and fromdissolution by naturally occurring lipids and surfactants. The latterare expected to have only limited penetration into the hydrogel, forexample, due to hydrophobicity, molecular weight, conformation,diffusion resistance, etc. In the case of a hydrophobic drug which haslimited solubility in the hydrogel matrix, the particulate form of thedrug may also serve as the release rate modifying agent. In oneembodiment, a microemulsion of a hydrophobic phase and an aqueoussolution of a water soluble molecular compound, such as a protein,peptide or other water soluble chemical is prepared. The emulsion is ofthe “water-in-oil” type (with oil as the continuous phase) as opposed toan “oil-in-water” system (where water is the continuous phase). Otheraspects of drug delivery are found in U.S. Pat. Nos. 6,632,457;6,379,373; and 6,514,534, each of which are hereby incorporated byreference, with the instant specification controlling in case ofconflict. Moreover, drug delivery schemes as described in U.S.2008/0187568 filed Feb. 6, 2008, which is hereby incorporated byreference herein (in case of conflict the present specificationcontrols), may also be used with the hydrogels herein.

Controlled rates of drug delivery also may be obtained with the systemdisclosed herein by degradable, covalent attachment of the bioactivemolecules to the crosslinked hydrogel network. The nature of thecovalent attachment can be controlled to enable control of the releaserate from hours to weeks or longer. By using a composite made fromlinkages with a range of hydrolysis times, a controlled release profilemay be extended for longer durations.

Fiducial Marking

An application for the hydrogels is use as a fiduciary marker. Fiduciarymarkers are used in a wide range of medical imaging applications.Different images of the same object may be correlated by placing afiduciary marker in the object. In radiotherapy, fiducial points aremarkers to facilitate correct targets for treatment. A radiation plan isdeveloped to administer desired radiation doses to a tumor target sitewith due consideration given to limiting exposure of other tissues.Plans may be developed through simulations. Plans relate to the exactarea that will be treated, the total radiation dose that will bedelivered to the tumor, how much dose will be allowed for the normaltissues around the tumor, and the safest paths for radiation delivery.The plans are typically developed using computers with suitablesoftware. Many checks should be made to ensure that the treatments arebeing delivered exactly as planned. The area selected for treatmentusually includes the whole tumor plus healthy tissue around the tumor;these are the treatment margins. Radiation can come from a machineoutside the body (external-beam radiation therapy) or from radioactivematerial placed in the body (brachytherapy).

Hydrogels as described herein may be used as fiduciary markers. Examples1 and 2 describe hydrogels used fiduciary markers. The hydrogel wassuccessfully used to completely fill lumpectomy cavities, partially fillcavities, and to mark cavities that were sutured closed. Examples 3 and4 describe radioopaque (RO) agents used in combination with hydrogels.The RO agents enhance contrast with the surrounding tissue. Flowableprecursors may be used to make the hydrogels in an iatrogenic site insitu. The precursors may be macromers, polymers, or monomers. Thehydrogels may be made to be low-swelling. In general, precursors may becombined as described herein at an iatrogenic site to make acovalently-crosslinked material, e.g., a hydrogel that adheres to themargins of the site and has a stable shape. In cases where thelumpectomy cavity walls are opposed with sutures, as in oncoplastyprocedures, the material can fill all the voids remaining in the cavity,still defining the cavity margins.

Embodiments thus include making a radiation plan using a hydrogel as afiduciary marker. The plan may be in written form or stored in acomputer readable medium. The term plan in this context refers to aproduct that can be exchanged in written or electronic form betweenpersons and excludes intentions or other mental processes. Such plansmay comprise a radiation dose or regimen and margin values. Similarly, arole as a fiducial marker may include imaging a site with a hydrogelrepeatedly over time and in combination with providing radiation to thesite. The site and marker may be imaged by a plurality of imagingdevices as are typically used in the medical arts.

The hydrogels may be provided in flowable form to the site, e.g., asflowable precursors. The precursors may be dissolved in, or suspendedin, a liquid and applied to the site. The precursors combine to form ahydrogel having a unitary continuous phase. Alternatively, the hydrogelsmay be provided as a plurality of particles that substantially contacteach other, with the hydrogel phase being discontinuous. The particlesmay be made to have a lubricity and maximum diameter for manual passageout of a syringe through a 3 to 5 French catheter, or a 10 to 30 gaugeneedle. Artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated.

The hydrogels may be used to substantially fill a site. Substantiallyfull means that the site is effectively full, with some allowances beingmade for elasticity of the site and packing of the hydrogel. Thehydrogels may also be used to partially fill a site, e.g., from about10% to about 90%; artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated.

A lumpectomy with a 3 cm diameter has a volume of about 14 cc, thusabout 14 ml of hydrogel would be required to completely fill the cavitywithout excessive tension. Accordingly, the volume of the material maybe tailored to the particular defect, e.g., from, about 1 ml to about100 ml; artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated, e.g., from6 ml to about 40 ml, or at least 5 ml. The implants tend to have avolume such that the implant includes at least one region withdimensions of more than 1×1×1 cm or more than 1×2×2 cm. Thus embodimentsinclude implants formed in situ with at least one region having threedimensions each in the range of 1 to 3 cm; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 1×1×2 cm, 1×2×2 cm, 3×2×1 cm. The regionmay contain a continuous phase of matrix or packed particles. Theseembodiments are in contrast to other tissue matrices or coatings. By wayof contrast, a layer of material that is only 5 mm thick would notcontain a 1×1×1 cm region. Useful material features include tissuecompatibility; the material is to be tissue compatible, with no systemictoxicity at high doses. Another feature is implant stability wherebydimensions do not appreciably change following implantation for apredetermined amount of time. Another feature is biodegradability: thehydrogel may gradually soften, liquefy and absorb after implantation.

Alternatively, the hydrogels may be fully or partially permanent and notbiodegradable, e.g., for cosmetic applications such as breast or facialsites. Standard lumpectomies may result in a compromised cosmeticresult, and often require whole breast irradiation due to imprecisetumor bed visualization.

Particles

One embodiment of the invention is directed to filling an iatrogenicsite with a collection of particles that are small, pliable, andslippery so that they flow easily into a site and its irregularities,pack closely, provide stability, optionally are biodegradable, andprovide good visualization of the margins.

One process for making particles involves creation of a matrix that isbroken up to make the particles. Thus matrices, and matrices made withprecursors as described herein, may be created and then broken up. Onetechnique involves preparing the hydrogel and grinding it, e.g., in aball mill or with a mortar and pestle. The matrix may be chopped ordiced with knives or wires. Or the matrix could be cut-up in a blender.Another process involves forcing the hydrogels through a mesh,collecting the fragments, and passing them through the same mesh oranother mesh until a desired size is reached.

The particles may be separated into collections with a desired sizerange and distribution of sizes by a variety of methods. Very finecontrol of sizing is available, with sizes ranging from 1 micron toseveral mm, and with a mean and range of particles sizes beingcontrollable with a narrow distribution. Artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated. About 10 to about 500 microns is one such rangethat is useful, with sizes falling throughout the range of having a meansizing at one value within the range, and a standard deviation centeredaround the mean value, e.g., from about 1% to about 100%. A simplemethod for sizing particles involves using custom-made or standardizedmesh sizes. In addition to standard U.S. and Tyler mesh sizes, sievesare also commonly used in the Market Grade, Mill Grade, and TensileBolting Cloth. Hydrogels forced through meshes have been observed toshow deformation so that the particle size is not precisely matched tomesh sizes; nonetheless, mesh sizes may be chosen to achieve a desired aparticle size range. A spheroidal particle refers to a particle whereinthe longest central axis (a straight line passing through the particle'sgeometric center) is no more than about twice the length of othercentral axes, with the particle being a literally spherical or having anirregular shape. A rod-shaped particle refers to a particle with alongitudinal central axis more than about twice the length of theshortest central axis.

Particles may also be made directly. In the case of ionic materials,well-controlled processes for making particles are known to artisans,for instance dropping small amounts of a polysaccharide into a bath ofions. Photopolymerization techniques are known for free radicalpolymerization, e.g., as in U.S. Pat. No. 5,410,016, which is herebyincorporated by reference herein; in case of conflict, the presentspecification controls. Emulsion-based techniques are also available. Inone method, hydrogel microspheres are formed from polymerizablemacromers or monomers by dispersion of a polymerizable phase in a secondimmiscible phase, wherein the polymerizable phase contains at least onecomponent required to initiate polymerization that leads to crosslinkingand the immiscible bulk phase contains another component required toinitiate crosslinking, along with a phase transfer agent. Additionally,a polymerizable phase, containing all components for reaction, but witha slow polymerization rate, can be introduced into a second immisciblephase where it is dispersed into microspheres prior to polymerization.The polymerization arts also provide for micellar and microemulsiontechniques for making particles.

A collection of microparticles may include sets of particles. Forinstance, some particles may be made to contain a radioopaque agent,with those particles forming a set within the collection. Other sets aredirected to particle sizes, with the sets having distinct shapes or sizedistributions. As discussed, particles can be made with well-controlledsizes and divided into various sets for combination into a collection.

Particles with radioopaque agents may be blended with particles that arefree of a radioopaque agent to make a collection of particles with adesired radiopacity. Example 3 details methods for making radioopaquehydrogels. The collection may thus have a percentage of iodine, forinstance an amount that is between about 0.05% and 5%; artisans willimmediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated, e.g., from about 0.1% toabout 0.4%. The iodine or other agent may be distributed between iodinecovalently bound to the particles and/or iodine mixed into the particles(e.g., iodine mixed into the particles at the time of formation), and/ormixed with the particles (e.g., added to a solution that contains theparticles). One or more radioopaque agents may be used to provide acollection with a target Hounsfield unit, e.g., more than about 50 or avalue between about 50 and about 2000; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., more than about 90, from 80 to 800.

Other sets are directed to degradability. One embodiment involves aplurality of sets each having a distinct degradability profile. Oneapplication is the use of a plurality of sets with distinctdegradability to promote tissue integration of an iatrogenic site. Thistechnique may be used to reduce changes in shape of the surroundingtissue by allowing gradual tissue ingrowth as the particles degrade. Oneproblem with iatrogenic sites is that they can contract or otherwisedeform the surrounding tissue. For instance, in breast cancer, theremoved tissue can cause a divot or otherwise poor cosmesis of thebreast. A collection of particles that exhibits staged degradation,however, provides for tissue to grow into the space over time andprovide a growth-filler. Some or all of the collection may be permanentand not degradable. Degradation times include 3 to 1000 days; artisanswill immediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated. For instance, a first set mayhave a median degradation time of from about 5 to about 8 days, a secondset a median time of from about 30 to about 90 days, and a third set amedian time of from about 180 to about 360 days.

The collections may optionally be prepared to be free of gas that maycause unwanted ultrasound echoes. One method involves simply degassingthe particles under vacuum, with and/or without a liquid solvent.Ultrasound tends to visualize particles more than about 20 microns,depending on the wavelength that is used. Size ranges for particlesinclude less than about 20 and less than about 20 microns.Alternatively, larger sized particles that have a high water content canalso avoid echogenicity. Accordingly, embodiments include particles thatessentially do not contribute to ultrasound images. In this context,essentially means that the particles do not interfere with thevisualization of other bodily features, even if it is possible tosometimes discern the presence of the particles by ultrasound.

Collections may be made with sizes and lubricity for manual injectionthrough a small gauge needle. Hydrophilic hydrogels crushed intospheroidal particles about 40 to about 100 microns diameter are smallenough to be manually injected through a 30 gauge needle.

Hydrophilic hydrogel particles were observed to pass with difficultythrough small gauge needles/catheters. The particle size contributes toresistance, as well as the viscosity of the solution. The particlestended to plug the needle. The resistance force is proportional to theviscosity of the fluid, with a more viscous fluid requiring more forceto push through a small opening.

It was unexpectedly found, however, that increasing the viscosity of thesolvent for the particles could lower the resistance to passage througha catheter and/or needle. This decrease may be attributed to using asolvent with a high osmolarity. Without being bound to a particulartheory, the addition of these agents to improve injectability was causedby particle shrinkage, increased free water between particles whichdecreased particle-to-particle contributions to viscosity, and increasedviscosity of the free water, which helped to pull the particles into andout of the syringes, preventing straining and plugging. The use of alinear polymer may further contribute thixotropic properties that areuseful to prevent settling and encourage movement of the particlestogether with the solvent, but exhibit shear thinning when being forcedout of a small opening. This approach was also observed to solve anotherproblem, namely, a difficulty in moving particles from a solutionthrough a needle/catheter since the particles tended to settle andotherwise elude pick-up. Expulsion through small bore openings ofsolutions of particles in aqueous solvent were observed; the solventtended to move preferentially out of the applicator, leaving an excessof particles behind that could not be cleared from the applicator, orthat plugged it, or in some instances could be cleared but only by useof an unsuitably large force not suited to an average user operating ahand-held syringe. The addition of osmotic agents, however, contributedviscosity and/or thixotropic behavior that helped to empty particlesfrom an applicator. Embodiments of the invention include the addition ofan osmotic agent to a plurality of particles. Examples of such agentsinclude salts and polymers. Embodiments include polymers, linearpolymers, and hydrophilic polymers, or combinations of the same.Embodiments include polymers of between about 500 and about 100,000molecular weight; artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated,e.g., about 5000 to about 50,000 molecular weight. Embodiments include,for example, a concentration of about 1% to about 50% w/w osmotic agent;artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., 10% to 30%.The agent and hydrogel may be introduced into a patient and may be partof a kit for the same.

Brachytherapy

Several brachytherapy techniques are used in cancer treatment. Inbrachytherapy, radioactive isotopes are sealed in pellets (seeds). Theseseeds are placed in patients. As the isotopes decay naturally, they giveoff radiation that damages nearby cancer cells. If left in place, aftera few weeks or months, the isotopes decay completely and no longer giveoff radiation. The seeds will not cause harm if they are left in thebody (permanent brachytherapy), although undesired migration from thesite of the implant has been observed Brachytherapy can be given as alow-dose-rate or a high-dose-rate treatment: In low-dose-rate treatment,cancer cells receive continuous low-dose radiation from the source overa period of several days.

A conventional approach is a high-dose-rate brachytherapy source inballoon catheter placed in a lumpectomy site. In other words, balloonsare placed into the cavity, and radioactive seeds are placed inside theballoon for discrete periods via a percutaneous attachment. Since thereare many possible volumes of tissue, the clinician would generallyselect from a menu of sizes and pick the one that seems to be about theright size for the site. The MAMMOSITE System (Hologic) can be insertedduring surgery, and allows for irradiation in 5 days of treatment.Negatives of this system include a high reported infection rate (12-16%)presumably due to the percutaneous access, patient issues such asdiscomfort and balloon rupture, and the need for extra equipment such asa high-dose-rate brachytherapy source, a shielded room, and otherspecialized equipment.

An embodiment of the invention is brachytherapy, with a radioactive seedor other source disposed with or inside a matrix in a cavity. The sourcemay be disposed in a bulk hydrogel that is continuous, in a hydrogelparticle, or mixed with hydrogel particles, or any combination thereof.The conformal positioning of the hydrogels provides significantadvantages for providing radiation where it is needed. Moreover, theparticles or matrices may be used in combination with MAMMOSITE or otherradiation sources. The source may be present at the time of placement,in a mixture with hydrogel precursors or particles, or placed after thematrix or hydrogel is placed.

Breast brachytherapy may only proceed if there is adequate space betweenthe balloon surface and the patient's skin. If inadequate distancebetween those surfaces is observed, the skin is sometimes damaged, orbreast brachytherapy is curtailed. A hydrogel as described herein may beinjected between the balloon surcease and the surrounding tissue and/orskin, effectively increasing that distance, allowing brachytherapy toproceed.

Tissue Augmentation

Hydrogels as set forth herein may be used for tissue augmentation. Theuse of collagen as for dermal augmentation is well known. Hydrogels, forexample particulates, may be used for dermal filler or for tissueaugmentation. Embodiments include injecting or otherwise placing aplurality of particles in a tissue, or forming a hydrogel in situ. Thematerial may be injected or otherwise placed at the intended site.

Spacers

Hydrogels as set forth herein may be used to separate tissues to reducea dose of radioactivity received by one of the tissues. As set forth inU.S. Pat. No. 7,744,913, which is hereby incorporated by referenceherein for all purposes with the present specification controlling incase of conflict, spacer materials may be placed in a patient. Certainembodiments are a method comprising introducing a spacer to a positionbetween a first tissue location and a second tissue location to increasea distance between the first tissue location and the second tissuelocation. Further, there may be a step of administering a dose ofradioactivity to at least the first tissue location or the second tissuelocation. A method, for example, is delivering a therapeutic dose ofradiation to a patient comprising introducing a biocompatible,biodegradable particulate hydrogel, e.g., a collection of particlesoptionally with radioopaque contents, between a first tissue locationand a second tissue location to increase a distance between the firsttissue location and the second tissue location, and treating the secondtissue location with the therapeutic dose of radiation so that thepresence of the filler device causes the first tissue location toreceive less of the dose of radioactivity compared to the amount of thedose of radioactivity the first tissue location would receive in theabsence of the spacer. The spacer may be introduced an injectablematerial and is a gel in the patient that is removed by biodegradationof the spacer in the patient. An example is the case wherein the firsttissue location is associated with the rectum and the second tissuelocation is associated with the prostate gland. The amount of reductionin radiation can vary. Embodiments include at least about 10% to about90%; artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., at leastabout 50%.

The radiation may alternatively be directed to a third tissue so thatthe first tissue or the second tissue received a lower amount ofradiation as a result of its separation from the other tissue(s). Thefirst tissue and the second tissue may be adjacent to each other in thebody, or may be separate from each other by other tissues.

Spacer volumes for separating tissues are dependent on the configurationof the tissues to be treated and the tissues to be separated from eachother. In many cases, a volume of about 20 cubic centimeters (cc's ormls) is suitable. In other embodiments, as little as about 1 cc might beneeded. Other volumes are in the range of about 5-1000 cc; artisans willimmediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated, e.g., 10-30 cc. In someembodiments, spacers are administered in two doses at different times soas to allow the tissues to stretch and accommodate the spacer andthereby receive a larger volumes of spacer than would otherwise bereadily possible. Tissues to be separated by a spacer include, forexample, at least one of a rectum, prostate, and breast, or a portionthereof. For instance, a first portion of a breast may be separated froma second portion.

Administration of Hydro Gels

One mode of administration is to apply a mixture of precursors and othermaterials (e.g., therapeutic agent, viscosifying agent, accelerator,initiator) through a needle, cannula, catheter, or hollow wire to aiatrogenic site. The mixture may be delivered, for instance, using amanually controlled syringe or mechanically controlled syringe, e.g., asyringe pump. Alternatively, a dual syringe or multiple-barreled syringeor multi-lumen system may be used to mix the precursors at or near thesite. Alternatively, a plurality of hydrogel particles may be appliedinstead of the precursors. Precursors and particles may also be mixed.

Either while the cavity is still open, or following oncoplasty andbefore skin closure, or after skin closure, a small gauge needle or apercutaneous catheter in the site can first be used to aspirate air orfluid, and then used to inject materials for the hydrogel, e.g.,hydrogel particles or an in situ curing material. Injection at or near(within a few days or a few weeks) the time of surgery is distinct from,and provides a different outcome than, using the materials in a locationthat has undergone healing processes for a significant time. In thiscontext, a few days includes 1 to 13 days and a few weeks includes 2 to10 weeks; artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated. Theprecursors may be chosen so that degradation products are absorbed intothe circulatory system and cleared from the body via renal filtration.One delivery embodiment is an applicator that consists of two syringesattached to a Y-connector with an integral static mixer. Atimplantation, precursors are injected from the syringes through a small,flexible catheter. Alternatively, a syringe of other application may beconnected to the catheter and used to provide particles of hydrogel. Theparticles may be fully hydrated, partially hydrated, or desiccated. Thecatheter may be left in the suture line at the time of surgery andextend to the iatrogenic site. The catheter is removed after thehydrogel is delivered.

Alternatively, a needle or catheter may be used to deliver the hydrogelafter the site is closed, optionally with indirect imaging for guidingthe distal tip of the applicator to the intended site.

Applicators may be used in combination with the matrices and/orprecursors. Kits or systems for making hydrogels may be prepared. Thekits are manufactured using medically acceptable conditions and containcomponents that have sterility, purity and preparation that ispharmaceutically acceptable. The kit may contain an applicator asappropriate, as well as instructions. A therapeutic agent may beincluded pre-mixed or available for mixing. Solvents/solutions may beprovided in the kit or separately, or the components may be pre-mixedwith the solvent. The kit may include syringes and/or needles for mixingand/or delivery. The kit or system may comprise components set forthherein.

One system uses a dual container applicator, e.g., double barreledsyringe, for delivering at least one precursor. One syringe may haveleast one precursor and the other syringe may have an activator foractivating the precursor, e.g., an initiator. Or each syringe may have aprecursor, with the precursors making a matrix as a result of mixing.

Another option for a kit or system is a collection of particles whereinat least some of the particles are dehydrated or are desiccated. Oneembodiment provides particles that are 30% to 100% desiccated; artisanswill immediately appreciate that all the ranges and values within theexplicitly stated ranges are contemplated. A kit may include one or moretherapeutic agents that may optionally be mixed with the particles. Forexample, the kit may have a first agent and a second agent that aremixed with a half-desiccated set of particles in a solution so that theparticles imbibe the solution and agent. A first set of particles may bemixed with a first agent and a second set with a second agent, or theagents may be mixed with a set of particles. The sets of particles withthe imbibed agents may be further mixed with other particles to make acollection for placement into a patient.

Other embodiments provide a single applicator, e.g., one syringe thatcomprises particles for delivery. One embodiment provides a containerfor particle delivery (e.g., syringe barrel, vial with septum) that doesnot require the addition of further contents, e.g., the particles areused neat, or are already in a solution or slurry that will be placedinto the patient. This allows for the use of injectable preformedhydrogel slurries, eliminating the need for reconstitution, multiplesyringes, and allowing for stop-and-start injections without fear ofneedle plugging. The particle solvent may be essentially water, meaningabout 99% v/v of the solvent is water, with salts or buffers beingpresent as desired. Other solvents may be used that are safe andbiocompatible, e.g., dimethylsulfoxide.

One method of treating a patient is directed to forming an implant thatclosely fills a cavity. The method relates to filling an iatrogenic sitewith at least one flowable material that flows into the site whilesubstantially replacing the volume and shape of the removed material asdetermined by visual observation as the filling is performed. Thematerial may comprise a collection of particles or a hydrogel precursor.

In some cases, a site may be revisited with a second procedure. This mayinvolve re-operating the iatrogenic site to remove the matrix,surgically removing additional tissue, and repeating the method to forma new hydrogel. In some of these cases, the original hydrogel comprisesa visually-observable visualization agent and removal of the hydrogelcomprises aspirating the site until no more of the visualization agentis observable to the naked eye in the aspirate, with the visualizationagent optionally being a dye, or a dye chosen from the group consistingof green and blue dyes.

EXAMPLES Example 1: Conformal Filling with Hydrogels

Under an approved protocol, three cadaver specimens were obtained forbilateral lumpectomies; in one, unilateral lumpectomy was performed dueto prior breast surgery. On the CT-simulation table, each specimen waspositioned for left sided lumpectomy (using wedges). Lumpectomiesranging from 31 to 70 cc were performed. Following lumpectomy, a 0.25″diameter silicone catheter was placed within the cavity, thesubcutaneous tissue was apposed, and the skin was closed. In one case,the superior and inferior cavity walls were apposed prior to closure.Before hydrogel injection, each underwent CT simulation (PhilipsBRILLIANCE BIG BORE CT, 3 mm slices, 120 kVp, 300 mA, 60 cm FOV).

Following CT simulation, 18 to 70 cc of hydrogel was injected within thecavity, the silicone catheter was withdrawn, and the hydrogel wasallowed to solidify. The hydrogel, when injected, has the viscosity ofwater but then, within 60 seconds, polymerized and formed a soft, solidgel. The hydrogel was DURASEAL, which is commercially available and isformed from a multi-armed PEG reacted with trilysine.

In three cases, the injected volume was equal to the lumpectomy volume(63, 70, and 35 cc). In the other two cases, only 18 cc of PEG-hydrogelwas injected (following 31 and 33 cc lumpectomies). CT-simulation wasrepeated. T2-weighted MR imaging was performed in the axial and sagittalplanes (Siemens ESPREE 1.5T MRI, turbo spin-echo, TR 5.0 sec, TE 106 10msec, FOV 26 cm, 3.0 mm slices, 256×256 matrix, 100% phase oversampling,130 Hz/pixel, echo-train length 17). Then cone-beam CT imaging wasperformed (Elekta Synergy, XVI software v.4.1b21, 1024×1024 flat-paneldetector, M10 collimator, BOWTIE filter, 120 kVp, nominal 40 mA/frame,nominal 40 ms/frame, 360° scan, 410×410×120 reconstruction, isotropic 1mm voxels). Finally, ultrasound imaging (7.1 MHz, B-K Medical 2101FALCON, #8658 4-9 MHz probe) was performed. After all imaging wascompleted, gross dissection was performed to confirm hydrogel locations(FIG. 3).

The hydrogel clearly defined the lumpectomy cavity on multiple imagingmodalities. An example from one lumpectomy procedure is shown (FIG. 3).On CT imaging (panel a), the homogeneous, water-density hydrogelcontrasted well with the lower density breast tissue. With T2-weightedMRI (panel b), the hydrogel was hyperintense and very prominent comparedwith the surrounding tissue. On T1-weighted imaging, the hydrogel had alow signal-intensity and was not as conspicuous as on T2-weightedimaging. The lumpectomy cavity was also visible on cone-beam CT imaging(panel c). A corresponding gross axial section (panel d; hydrogel dyedblue to improve visualization for this study) showed very similarfeatures as all three cross-sectional imaging modalities (note the flapof fat within the lumpectomy cavity). While ultrasound did not show asmuch cavity detail, the echolucent hydrogel contrasted well with thesurrounding breast tissue (panel e).

The hydrogel was successfully used to completely fill lumpectomycavities, partially fill cavities, and to mark cavities that weresutured closed. In three of the lumpectomy procedures, the cavity wasfilled with a volume of hydrogel equivalent to the volume of extractedtissue. For example, in FIGS. 3a and 3b , a 63 cc lumpectomy wasperformed and an equal volume of hydrogel was injected. The hydrogelcompletely filled the cavity and restored a normal, convex breastcontour. However, in two cases, a smaller volume of hydrogel wasinjected (simply marking the cavity, rather than filling and expandingit). In FIGS. 3c and 3d , a 31 cc lumpectomy was injected with only 18cc of hydrogel. While the hydrogel clearly marked the lumpectomy site,the cavity and breast surface remained concave. In one case, a 33 cclumpectomy was performed and then (as is the preference of some breastsurgeons), the superior and inferior walls of the cavity were suturedtogether. 18 cc of hydrogel was injected into this cavity, which markedthe edges of the cavity, outlining the apposed tissue (FIGS. 3e and 3f). In each situation, the hydrogel still clearly defined the cavitylocation on both CT and MR imaging. In all 5 cases, gross dissectionconfirmed that the full extent of the cavity was marked by hydrogel.

Example 2: Radiation Exposure Control with Conformal Hydrogels

The specimens of Example 1 had radiation plans developed forpre-hydrogel implant cases and post-hydrogel implant cases. Thepre-hydrogel and post-hydrogel CT scans were imported into a Pinnacletreatment planning system (v8.0m, Philips Radiation Oncology Systems).For first sets of plans, standard margin expansions (a 15 mm GTV-CTVexpansion and a 10 mm CTV-PTV expansion) was used for all fivepre-hydrogel and post-hydrogel plans (per the NSABP-B-39/RTOG-0413protocol). For a second set of plans, standard margins were used for theprehydrogel plans, but reduced margins (a 10 mm GTV-CTV expansion and a5 mm CTV-PTV expansion) were used for the post-hydrogel plans.

Intensity modulated radiotherapy (IMRT) treatment planning was performedusing five, non-coplanar beams designed to minimize normal structureradiation exposure. Non-target structures included the ipsilateral andcontralateral lungs, the heart, and the ipsilateral breast tissue notincluded in the PTV (breastNotPTV). Ipsilateral breast tissue wasdefined as that tissue lying within standard, whole-breast tangent beams(per the NSABP-B-39/RTOG-0413 protocol). Appropriate dose-volumeobjectives were assigned to these structures, and plans were consideredacceptable when the following constraints were achieved: breastNotPTVV50%<50%, ipsilateral lung V30%<15%, and heart V5%<40%.

When using standard margin expansions, the hydrogel tended to increasenormal tissue radiation doses. Five-field, partial-breast radiationtreatment plans were generated for each of the five lumpectomyprocedures; one plan was generated before hydrogel injection and asecond plan was generated after hydrogel injection. As expected, boththe lumpectomy cavity and the PTV were larger after hydrogel placementand normal tissue doses were modestly increased. With hydrogelinjection, mean cavity volume increased from 15.7 to 41.4 cc, a changeof 25.7 cc (95% confidence interval 7.8 to 43.7 cc). The mean PTV volumeincreased from 471.9 to 562.7 cc, a change of 90.8 cc (95% confidenceinterval 26.3 to 155.2 cc). While the mean cavity volume almost tripled,the fractional increase in the PTV size was much more modest (increasingonly 19%). As seen in FIG. 5, the hydrogel tended to expand the cavityoutward, away from the chest wall, but not laterally. As the CTVexpansion is limited to remain within the breast tissue, there wastherefore little net effect on the PTV size. Normal-tissue dosimetricparameters from all 5 lumpectomies are presented in FIG. 6; overall,when using standard treatment margins (25 mm), the hydrogel tended toincrease normal tissue doses. The breast (non-PTV) V50% increased in 4of 5 cases; the mean increase was 1.4% (95% confidence interval −1.7% to4.5%). This increase was modest compared with the volume constraint of50%. The ipsilateral lung V30% also increased in 4 of 5 cases. Theincreases were more sizable relative to the volume constraint of 15%(mean increase was 1.7%, 95% confidence interval −0.4% to 3.8%). Asanticipated, the deeper, larger cavities resulted in higher lung doses;in one case, the post-hydrogel plan reached the ipsilateral lung V30%limit of 15%. For all three left sided lumpectomies, the hydrogelincreased the heart V5%. But, in all cases, the volumes remained wellunder the 40% constraint (mean increase 3.1%, 95% confidence interval−3.0% to 9.2%).

When using reduced margin expansions, the hydrogel tended to decreasenormal tissue radiation doses. The reduced margins were made feasible bythe hydrogel's improved cavity visibility. As the hydrogel improvesvisualization of the lumpectomy cavity, its impact on normal-tissuedoses was also examined when smaller margin expansions were employed.Reduced margins may be appropriate due to reduced uncertainty in targetdefinition and also reduced day-to-day target localization error. Withreduced treatment margins (a 10 mm GTV-CTV expansion and a 5 mm CTV-PTVexpansion), the hydrogel tended to decrease normal tissue doses despitethe increase in lumpectomy cavity volume (compared with no hydrogel andstandard, 25 mm margins) (FIG. 7). The breast (non PTV) V50% decreasedin all five cases (mean change −3.2%, 95% confidence interval −6.4% to0.0%). The ipsilateral lung V30% also decreased in 4 of 5 cases (meanchange 1.5%, 95% confidence interval −4.1% to 1.1%). For all left-sidedlumpectomies, the heart V5% showed small decreases (mean change −0.8%,95% confidence interval −2.3% to 0.8%).

Example 3: Radioopaque Hydrogels and Imaging

The radiopacity of serial diluted Iohexol (OMNIPAQUE) was measured toobtain a CT number as a function of iodine concentration. A CT number(also referred to as a Hounsfield unit or number) is the densityassigned to a voxel in a CT (computed tomography) scan on an arbitraryscale on which air has a density −1000; water, 0; and compact bone+1000. The CT number of diluted Iohexol ranged from 2976 at a 50%concentration, to 37 at a 0.2% concentration. These corresponded to aplot of iodine concentration versus CT number, with an iodineconcentration of approximately 0.15% resulting in a CT number of about90 (FIG. 8). Two different matrix formulations containing iodine atdifferent concentrations were tested. First, iodine with succinimidylglutarate (SG or SGA) functional terminal groups were complexed to 5000Dalton linear PEG to make a PEG molecule complexed with iodine (PEG-I).Second, potassium iodide (KI) was incorporated into the gel at differentconcentrations.

Iodine Incorporation into the PEG Molecule

A PEG SG (with an SG count of 2.3 per molecule) containing an iodinecore was synthesized. The PEG-I molecule was 6400 Daltons, of whichiodine was 381 Daltons (5.9%). Thus, for example, with this iodinecontent, the percent solids of PEG-I in hydrogel that resulted in 0.1%and 0.2% iodine concentration in the resultant matrix was 1.68 and3.36%. Table II shows how PEG-I concentrations can be manipulated toobtain a percentage iodine content, which in turn can be related to a CTnumber.

TABLE II The percent solids of PEG-I in hydrogel, and the correspondingpercent iodine concentrations. % iodine % PEG-I in gel 0.1 1.68 0.2 3.360.4 6.72 0.8 13.45

During sample preparation it was noted that the samples with high PEG-Iconcentrations gelled slowly, or not at all (Table III). This wasobserved at PEG-I concentrations up to about 20%. The potential reasonsfor this include crosslink interference due to SG end group proximity tothe iodine hydrophobic core, rapid hydrolysis of the end groups prior topolymerization, or the formation of micelles due to the hydrophobicregion, that may or may not have polymerized.

TABLE III Hydrogel samples evaluated *. ID Description Condition 1 0.1%I, 1.68% PEG-I, 13.32% 4a20kSGA Gel 2 0.2% I, 3.36% PEG-I, 11.64%4a20kSGA Gel 4 0.8% I, 13.45% PEG-I, 1.55% 4a20kSGA Liquid 5 0.1% I, 10%4a20kSGA, 0.120% KI Gel 6 0.2% I, 10% 4a20kSGA, 0.241% KI Gel 7 0.4% I,10% 4a20kSGA, 0.481% KI Gel 8 0.8% I, 10% 4a20kSGA, 0.963% KI Gel 9 4%10 μm MSs, 10% 4a20kSGA Gel * PEG-I refers to a PEG with a covalentlyattached iodine; MSs refers to microspheres; branched PEG molecules aredescribed as NaXXkYYY, with N being the number of arms, XX being the MWin thousands, and YYY indicating the functional group at the arms'termini for those arms not terminating in an iodinated groups. Thus4a20kSGA refers to a 4-armed PEG of approximately 20,000 MW with SGAtermini.Free Iodine Incorporation into the Gel

Potassium iodide (KI) was also loaded into certain hydrogels. KI is231.3 Daltons, and iodine is 192.2 Daltons (83.1%) so that theconcentration of KI required to obtain the same iodine concentrationscould be calculated. 0.1% iodine is 0.120% KI, 0.2% iodine is 0.241% KI,and 0.8% iodine is 0.963% KI.

Microsphere Incorporation into the Gel

A 4% microsphere loading was used. When loaded into microspheres at aconcentration of 20%, then the iodine concentration in hydrogel, given a4% microsphere loading in the hydrogel, is about 0.8%. The microsphereswere made as described above.

Methods

Non-sterile rods of gel with either 0.1, 0.2, 0.4 or 0.8% incorporatediodine were created by injecting 5 ml of in situ gelling polymer into 10ml syringes, creating plugs approximately 13.5 mm diameter and 30 mmlength. Conditions were controlled to prevent gel hydrolysis prior totesting.

Gel samples underwent computed tomography imaging while still insyringes such that CT number was determined. Syringes were placed on theCT couch (long axis of the gel samples aligned with the couch). Thefollowing CT scanner and scan settings were used: Philips BRILLIANCE BIGBORE CT simulator, slice thickness 3 mm, 120 kVp, 300 mA, FOV 60 cm.Gels were removed from syringes by blowing them out with air from a 20ml syringe. Following removal the gel plugs were weighed. Followingimaging samples were placed in 150 ml containers, each containing 100 mlof PBS, and stored at room temperature prior to additional testing.

CT Imaging

CT Imaging showed a difference in radiopacity. As shown n FIG. 8, bothiodine loading methods produced similar results, with a linear HUresponse to iodine concentration. These data have a similar slope tothat obtained earlier with Iohexol (OMNIPAQUE), although there was aslight offset.

Example 4: Radiopacity of Hydrogel with Bound Iodine

This example describes the radiopacity of different levels of triodoobenzoate (TIB) loading, along with different hydrogel percent solids.The evaluated materials and their estimated (based on Example 1)Hounsfield Units (HU) are shown in Table VI. Hydrogel plugs were createdinside silicone tubing of 0.375 inch ID and placed in conical tubes toprevent evaporation. An 8a20kSGA PEG with 3-4 terminal TIB (31%substitution) or about 5 terminal TIB (61% substitution) was reactedwith trilysine to make the gels. A neutral hydrogel pH was used preventexcess hydrolysis prior to testing. There was fairly good agreementbetween the estimated radiopacity and that actually measured at timezero.

TABLE VI The four different hydrogels evaluated, along with their iodineconcentration [I] and radiopacity (HU). % of PEG arms with TIBattachments 31% TIB substitution 61% TIB substitution 5% Solids hydrogelHydrogel [I]: 0.21% Hydrogel [I]: 0.35% Estimated HU: 100 Estimated HU:155 10% Solids hydrogel Hydrogel [I]: 0.42% Hydrogel [I]: 0.70%Estimated HU: 180 Estimated HU: 280

Following the initial radiopacity measurements, samples were taken fromthe syringes and placed in conical tubes containing tap water. Sampleswere stored at room temperature (RT), and at each time point the sampleswere removed from the vials, weighed, and rescanned. The radiopacity(RO) over time, without correction for swelling, is shown in FIG. 9. Theradiopacity corrected for swelling is shown in FIG. 10. This datademonstrates some important features. First, the sample swellingdemonstrates ongoing hydrolysis, showing that the tested formulationwill eventually liquefy, and if implanted, will absorb. Second, whencorrected for swelling, the data demonstrates that the iodine isremaining bound to the precursor. This latter observation shows thatradiopacity may be maintained throughout the lifetime of the implant ifdesired.

Example 5: Osmotic Agents for Injectable Slurries

The addition of osmotic agents was observed to reduce the force requiredto move the particles through a small opening. The use of linearpolymers contributed viscosity and, without being bound to a particulartheory, a thixotropic effect. FIG. 11A is a plot of results of a slurryinjection force testing trial. Solutions of covalently crosslinkedmultiarmed PEG hydrogel particles of about 70 micron diameter wereformulated in 28% free water with different concentrations of linear PEG(20 k molecular weight). Materials were injected using a 3 cc syringeand 18 gauge 15 cm needle, with the force being monitored and reportedin N.

FIG. 11B shows shrinkage in solutions of PEG. Covalently crosslinkedmultiarmed PEG hydrogel plugs were made as described and exposed for 24hrs in 37° C. phosphate buffered solution (PBS, a physiological saline)containing 20 k linear PEG at different concentrations as indicated. Theplugs were observed to shrink (negative swelling), and 20% PEG solutioncaused about 70% shrinkage.

Further Disclosure

An embodiment of the invention is a pharmaceutically acceptable implantsystem or kit comprising a collection of pharmaceutically acceptable,covalently-crosslinked hydrogel particles having a radioopaque agentcovalently attached to a plurality of the particles in the collection,with the radioopaque agent being present in the collection at aconcentration of at least about 0.1% w/w. Another embodiment is aprocess for making an implantable system comprising preparing a hydrogelmatrix comprising covalently attached radioopaque agents and breakingthe matrix into a collection of pharmaceutically acceptable,covalently-crosslinked hydrogel particles. Another embodiment is apharmaceutically acceptable implant system or kit comprising acollection of pharmaceutically acceptable, covalently-crosslinkedhydrogel particles that comprises a plurality of sets of the particles,with the sets having different rates of biodegradation. Anotherembodiment is a method of treating a patient with a pharmaceuticallyacceptable implant system comprising implanting a collection ofpharmaceutically acceptable, covalently-crosslinked hydrogel particles.Another embodiment is a method for treating a tissue comprising placinga hydrogel in an iatrogenic site, wherein the hydrogel conforms tomargins of the site and has a Hounsfield number of more than about 50.Another embodiment is a plurality of, or collection of, particles foruse as: an implant, a spacer, a fiduciary marker, or an implant for aiatrogenic site.

These methods, processes, collections, hydrogels, particles, and systemsmay comprise, for example, one or more of the following features: theparticle collection further comprising particles free of acovalently-bound radioopaque agent; the collection further comprising anon-covalently bound radioopaque agent; wherein the collection particlesare spheroidal with a maximum diameter of between about 20 to about 200microns, with the particles being biodegradable to produce onlydegradation products that are absorbed into the circulatory system andcleared from the body via renal filtration; with the particles beinghydrolytically biodegradable; wherein the particles, before hydrolysis,have a total swellability in physiological solution of no more thanabout 30% by volume; wherein the degradation products comprise apolyethylene glycol covalently bound to the radioopaque agent, with theradioopaque agent comprising iodine; wherein the polyethylene glycol isa branched polyethylene glycol with at least four arms; wherein between25% and 90% of the arms comprise the radioopaque agent; with thecollection having a lubricity and maximum diameter for manual passageout of a syringe through a 30 gauge needle; further comprising anosmotic agent that comprises a linear hydrophilic polymer, with theagent present in a mixture with the collection; wherein the collectionof particles is completely biodegradable at a time between about 30 andabout 365 days; wherein the collection comprises a plurality of sets ofthe particles, with the sets having different rates of biodegradation;wherein a first set of the particles is biodegradable within about 8 toabout 12 days and a second set of the particles is degradable withinabout 45 to about 55 days; wherein the particles are hydrolyticallydegradable; further comprising an applicator, with the particles beingdisposed in the applicator; wherein the particles are dehydrated;further comprising a container of physiological saline fluidlyconnectable to the applicator to mix the saline and particles in theapplicator; further comprising a therapeutic agent; further comprising aradiation source; wherein the spacer or matrix is formed from a firstprecursor comprising a plurality of first functional groups and a secondprecursor comprising a plurality of second functional groups, with thefirst functional groups forming covalent bonds with the secondfunctional groups to thereby form the matrix; wherein at least oneprecursor further comprises the radioopaque agent; wherein the particlesare prepared by grinding, milling, chopping, micellar polymerization, oremulsion polymerization; wherein a first set of the particles isbiodegradable within about 8 to about 12 days and a second set of theparticles is degradable within about 45 to about 55 days; a set ofparticles that is biodegradable within about 60 to about 90 days;wherein the particles are hydrolytically degradable; comprising aplurality of the particles having a covalently attached radioopaqueagent, with the radioopaque agent being present in the collection at aconcentration of at least about 0.1% w/w; wherein the particles areformed from a first precursor comprising a plurality of first functionalgroups and a second precursor comprising a plurality of secondfunctional groups, with the first functional groups forming covalentbonds with the second functional groups to thereby form the matrix, withat least one of the precursors comprising polyethylene glycol; whereinat least one of the precursors comprises a polyethylene glycol having aplurality of branches terminated with triiodobenzoate; a plurality ofthe particles having a covalently attached radioopaque agent, with theradioopaque agent being present in the collection at a concentration ofat least about 0.1% w/w; placing the collection between two tissues andpreparing a radiation treatment plan that comprises a therapeutic doseof radiation to treat a cancer in one of the tissues; comprising placingthe collection in a tissue for augmentation; with the hydrogel furthercomprising a radioopaque agent; introducing a liquid comprising ahydrogel precursor into the site that flows into the site and reacts inthe site to form the hydrogel as a covalently crosslinked continuousphase that adheres to the margins; comprising a second precursor thatreacts with the first precursor to form covalent bonds to form thehydrogel; wherein the precursor comprises a covalently bound radioopaqueagent; wherein the radioopaque agent comprises iodine; wherein theprecursor comprises a branched polyethylene glycol, with the radioopaqueagent being disposed on at least one of the branches; further comprisingsubstantially filling the site with the hydrogel; wherein the hydrogelis biodegradable; forming a radiation plan based on the hydrogel as afiducial marker; wherein the plan sets forth margins of less than about20 mm; wherein the hydrogel comprises a collection ofcovalently-crosslinked hydrogel particles; wherein the a collection ofhydrogel particles comprises a radioopaque agent covalently attached toa plurality of the particles in the collection, with the radioopaqueagent being present in the collection at a concentration of at leastabout 0.1% w/w.

Various embodiments of the invention have been set forth herein. Ingeneral, features of the various embodiments may be mixed and matchedfor further combinations that are not explicitly detailed. Headings areset forth only for organizational purposes and do not limit the scope ofthe disclosure.

1.-15. (canceled)
 16. A method for radiation therapy comprisingintroducing a hydrogel spacer at a site between a first tissue locationand a second tissue location to increase a distance between the firsttissue location and the second tissue location, the site being chosen todecrease radiation at the first tissue when the second tissue receives adose of therapeutic radiation, wherein the hydrogel spacer comprises apolysaccharide and a radioopaque agent.
 17. The method of claim 16,wherein the polysaccharide is hyaluronic acid.
 18. The method of claim16, wherein the radioopaque agent is a covalently attached radiopaqueiodine agent.
 19. The method of claim 16, wherein the first tissuelocation is associated with the rectum and the second tissue location isassociated with the prostate gland.
 20. The method of claim 16, whereinthe spacer provides a fiducial marker for administration of thetherapeutic radiation.
 21. The method of claim 20, further comprisingvisualizing the interface of the fiducial marker and the second tissue.22. The method of claim 21, wherein the visualizing comprisescomputerized tomography or X-ray.
 23. The method of claim 16, furthercomprising making a radiation plan based on a visualization of thehydrogel spacer, with the hydrogel spacer being in contact with thesecond tissue.
 24. The method of claim 23, with the plan having areduced uncertainty in target definition relative to a radiation planmade without the hydrogel spacer.
 25. The method of claim 16, whereinthe hydrogel spacer has a Hounsfield number of more than about
 50. 26.The method of claim 16, wherein the hydrogel spacer, as placed in thetissue, has a volume between 1 and 40 ml.
 27. The method of claim 16,further comprising visualizing margins of the hydrogel spacer with amachine selected from computerized tomography or X-ray.
 28. The methodof claim 16, further comprising a therapeutic agent in the hydrogelspacer.
 29. The method of claim 16, with the radiopaque agent beingpresent in the hydrogel spacer at a concentration of at least about 0.1%w/w.
 30. A method for radiation therapy comprising introducing ahydrogel spacer at a site between a first tissue location and a secondtissue location, the site being chosen to decrease radiation at thefirst tissue when the second tissue receives a dose of therapeuticradiation, wherein the hydrogel spacer comprises hyaluronic acid and acovalently attached radiopaque agent.
 31. The method of claim 30, hereinthe first tissue location is associated with the rectum and the secondtissue location is associated with the prostate gland.
 32. The method ofclaim 30, wherein the hydrogel spacer, as placed in the tissue, has avolume between 1 and 40 ml.
 33. The method of claim 30, wherein thehydrogel spacer has a Hounsfield number of more than about
 50. 34. Themethod of claim 30, wherein the spacer provides a fiducial marker foradministration of the therapeutic radiation.
 35. A method for radiationtherapy comprising (a) introducing a hydrogel spacer at a site between afirst tissue location and a second tissue location, the site beingchosen to decrease radiation at the first tissue when the second tissuereceives a dose of therapeutic radiation, wherein the hydrogel spacercomprises hyaluronic acid and a radiopaque agent and (b) visualizingmargins of the hydrogel spacer with external imaging.